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DETECTION OF ALLELIC EXPRESSION UTILIZING RNA FLUORESCENT IN SITU HYBRIDIZATION IN
MOUSE EMBRYONIC CELLS
Monica Antenos
A thesis submitted in conformity with the requirements for the Degree of Master of Science, Graduate Department of Zoology,
University of Toronto
8 Copyright by Monica Antenos 1998
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DETECTION OF ALLELIC EXPRESSiON UTILIZING RNA FLUORESCENT IN SITU HYBRIDIZATION
IN MOUSE EMBRYONIC CELLS
Monica Antenos, Master of Science, 1998
Graduate Department of Zoology, University of Toronto, Toronto, Ontario, Canada
ABSTRACT
Genornic imprinting in mammals is defined as a reversible epigenetic process which
renders the two parental genomes functiondly nonequivalent in the developing ernbryo.
Monoallelic expression is observed exclusively in seventeen imprinted genes identified to date,
however the time at which transcriptional silencing of one allele occurs is variable throughout
development and dependent on the gene in question. RNA fluorescent in situ hybridization
is utilised in this study as a rnethod of detecting when one allele of an imprinted gene undergoes
transcriptional silencing in the embryonic development of the mouse. The aim of this work is to
utilize this sensitive FISH technique, which targets the nascent transcripts of genes. in order to
visualise transcriptionally active alleles. Examination of the levels of active transcription in each
individual ce11 in a spectrum of tissues will reveal if al1 nuclei act synonymously and determine at
what embryonic stage an imprinted locus is silenced. This thesis attempts to optimize the
conditions for RNA FISH in two models - cultured ce11 lines and embryonic tissues of the mouse.
1 would Iike to thank Dr. Sue Varmuza who has given me the opportunity to pursue
graduate studies in her laboratory and for her guidance and support throughout the length of my
degree.
Many special thanks go to Andrea Jurisicova, Ian Rogers, Leancira Oppedisano and Dr.
MeUissa Mann for all their help and support; for m a h g lab iife very enjoyable, and for their "deep
thoughts" regarding Me's great mysteries over numerous and much needed c o f k breaks.
As well, I would like to thank Dr. Barbara Panning for her help and attention, as well as
her numerous suggestions which helped me to achieve the positive resdts presented here.
I would also like to thank Dr. Umberto De Boni and Paul Park for taking tirne out of their
busy schedules to answer numerous questions and offer suggestions. Their passion for studies of
the interphase nucleus was very contagious.
Special thanks also to Dr. Yoshio Masui for his guidance and suggestions made throughout
the length of this project.
And lastly, 1 would like to thank Ms Michelle Nola who has helped me through the most
difficult moments of this degree and who has helped me look forward and find an almost
extinguished light at the end of that proverbial tunnel. 1 am forever gratefd.
This work is dedicated to Rambo.
..* 111
TABLE OF CONTEXTS
CONTENT
Abstract
Table of Contents
List of Figures
List of Tables
List of Abbreviaîions
CXAPTER 1: INTRODUCTION
1 .1 General airn of this investigation
1.2 Genomic Imprinting 1.2.1 Identification of Imprinted Genes 1.2.2 Models of Imprinting 1.2.3 Allele specific patterns of expression of irnprinted genes
1.3 Dynarnics of the Nucleus 1.3.1 The interphase nucleus 1.3.2 Nuclear speckles 1.3.3 Nuclear tracks
1.4 Rationale
1.5 Objectives
CHAPTER2: MATERIALS AND METBODS
2.1 MATERIALS 2.1.1 Animais 2.1.2 DNA utilized as probes 2.1.3 Solutions, Glassware and Plasticware
PAGE
. . Il
.a.
Ill
2.2 MErnODS 22.1 Retrievd of mouse embryos
2.2.1.1 Superovulation of femaies for retrieval of mouse blastocysts
2.2.1.2 Dissection of pst implantation embryos 2.2.1.3 Retrievd of nuciei 22.1.4 Storage ofnuclei
22.2 Tissue culture 2.2.2.1 Mouse fibroblast cell line 2.2.2.2 Culturing of post implantation ernb~yos 2.2.2.3 Culturing of blastocysts and ectoplacental cones for
outgrowths 2.2.2.4 Embryos Squashes
2.2.3 FISH Probes 2.2.3.1 Nick translation of probes 22.3 -2 Probe preparation
2.2.4 FISHing 2.2.4.1 Dehydration of slides 2.2.4.2 Application of prepared probes
2.2.5 Detection of nascent trancnpts 2.2.5.1 Removal of excess non-specific binding of probe 2.2.5.2 Blocking and detection with fluorochrome 2.2.5.3 Counterstainuig and viewing of slides
3.1 Embryonic fibroblast cell line
3.2 Analysis of &y 7.5 embryonic nuclei utilin'ng RNA FISH
3.3 Increasing accessibility into the nucleus
3.4 Mouse embryonic tissues 3 -4.1 Embryos dissociated k e of trypsin 3 A.2 Blastocyst outgrowths and ectoplacental outgrowths 3.4.3 Squashed embryos
CaAPTER 4: DISCUSSION
4.1 Success of RNA FISH on cultured fïbroblast cells
4.2 Mouse embryonic cells 4.2.1 Recovery of nuclei 4.2.2 Cell types 4.2.3 Penetration of the nuclear membrane 4.2.4 Probes and target sequences
4.3 Blastocyst Outgrowths and Ectoplacentd ûutgrowths
4.4 Methods of improving the RNA FISH technique
4.5 Monoallelic expression of imprinted genes
4.6 The interphase nucleus and imprinted genes
CONCLUSIONS
REFERENCES
LIST OF FIGURES
FIGURES
1.
CONTENT PAGE
Autosomal chromosome imprkting map of the mouse
Verification of probe fr-agment size and biotin incorporation
Biallelic expression of fibronectin in fïbroblasts
Fibroblast cell culture r d t s
Fibrobiasts treated after fixation with triton X-100 or proteinase K
Prolonged treatments of triton X-100 after kation
Variations in 0.5% triton X-1 O0 treatment to examine biaiielic expression of fibronectin 35
Variations in 0.5% triton X- f O0 treatment to examine biallelic expression of fibronectin (fixative included CPC) 36
Mouse embryonic nuclei hybridized with Hl 9 and p-actin probes 3 9-40
Increasing accessibilty into the nucleus using proteinase K 43-44
RNA FISH on txypsin-free treated embryonic cells 46-47
II .
Blastocyst outgrowths examined by RNA FISH 49-50
Ectoplacental cone outgrowths examined by RNA FISH
vii
Squashed embryos examined by RNA FISH
LIST OF TABLES
TABLES COl?mENT PAGE
1. Imprinted genes and chromosome regions in 2 maxnmais
2. Fibroblast ce11 culture results for RNA FISH 28
3. Treatments aimed at increasing accessibility 41 into the nucleus
4. Trypsin-ke derived embryonic cek 51
ABBREVIATIONS
AS
bp
BSA
cDNA
CLSM
CO2
CPC
CSK
DAPI
depc
~ P C
EDTA
epc
ES cells
EtBr
exe
FISH
FITC
HCG
HCI
hnRNP
Igf2
Igf2r
Ins 2
ru kb
KSOM
mg
Angelman Syndrome
base pairs
bovine senun albumin
complernentaq deoxyribonucleic acid
confocal laser scanning microscope
carbon dioxide
cetylpyridinium chlonde
cytoskeletai b a e r
4' ,6-diamidino-2-phenylindole
dietyhl pynxarbonate
days p s t coitwn
ethylenediaminetetra-=tic acid disodium salt
embryonic ectoderm
ectoplacentai cone
embryonic stem cells
ethidium bromide
extraembryonic ectodenn
fluorescent in situ hybrid'ion
fluorescein isothiocyanate
hurnan chorionic gonadotrophin
hydrochloric acid
heteronuclear ribonucleoprotein particle
insulin-like growth factor 2
insului-like growth factor-2 receptor
Insuiin 2
international units
kilobases
potassium-mMed synthetic oviductal medium
rnilligram
X
kb
KSOM
ml
mM
mRNA
mRNP
NaCl
PBS
PCR
PMS
pre-mRNA
Pro k
PWS
RNA
RNAase A
snRNP
S S C
tRNA
ug
ul
U M
VRC
Xist
kilobases
potassium-modined synthetic oviductal medium
rnilligram
magnesiun chloride
rnilliliter
millimolar
messanger ribonucleic acid
rnessanger nbonuclear protein paxticle
sodium chloride
phosphate buffer saline
polymerase chah reaction
pregnant mare semm
pre-messenger ribonucleic acid
proteinase K
Prader-Willi Syndrome
ribonucleic acid
ribonuclease A
small nuclear ribonucleoprotein particle
sait-saturateci citrate
transfer ribonucleic acid
microgram
rnicroliter
micromolar
vanadyl ribonucleoside complex
X inactive specific transcript
CHAPTER 1
INTRODUCTION
1.1 GENERAL AIM OF THIS INVESTIGATION
The phenornenon of genomic imprinting in mammals is defined as an epigenetic marking of
the parental alleles of imprinted genes which modifies the genome differently in the male and
female germline leading to differential activity of the parental genomes in the offspring (Solter,
1988; Barlow, 1994; Nakao and Sasaki, 1996). Monoailelic expression of imprinted genes is
observed exclusively in seventeen genes to date (Table 1 ; John and Surani. 1996); however the
time at which transcriptionai silencing of one allele occurs is variable throughout developrnent and
dependent on the gene in question. Numerous studies undertaken by many investigators have k e n
incapable of determining at what stage one ailele is silenced and more importantly how that d e l e is
silenced. The primary goal of this thesis is to examine the developrnental stages at which an
imprinted gene is transcriptionally silenced using RNA Fluorescent in situ hybridisation (FISH) . Active transcription of a gene c m be visuaiised utilising RNA FISH whose target is the
pre-mRNA, or the nascent transcript, of a newly transcribed gene prior to its processing in the
nucleus. The use of such a sensitive technique allows for the detection and enurneration of sites of
transcription and should allow the investigator to see how many alleles are currently active in the
nucleus at the time of fixation. Theoretically, on a ce11 by ce11 basis. a specific tissue or an entire
embryo can be examined using this technique to determine if the silencing mechanism is operating
strictly in al1 nuclei examined, or to show whether the silencing mechanism is "leaky" and in what
ce11 types. Biallelic expression has been reported for a nurnber of imprinted genes at early
embryonic stages (Szabo and Mann, 1995b; Latham et al., 1994; and Mann et al.. 1995). RNA
FISH was to be utilised in this investigation to target the time at which an imprinted gene is
silenced during the developmental stages of mouse embryogenesis. This work has been aimed at
optimising the conditions for RNA FISH when applied to a number of different nuclei which
include both cultured cells as well as embryonic cells.
Li:
1.2 GENOMIC IMPIUNTING
1.2.1 Identification of Imprinted Genes
Contributions to the developing embryo corne from the genetic information passed on by
both the matemal and patemal genomes. However, studies in 1984 have clearly illustrated that the
parental genomes, although containing equivalent genetic information, do not contribute
functionally equivalent genetic information (Surani et al., 1984; McGrath and Solter, 1984).
Mouse embryos which contain either two matemal genomes (parthenotedgynogenotes) or two
patemai genomes (androgenotes) fail to deveiop normaiiy and resuit in characteristic developmentai
failures. Gynogenetic embryos, produced by the nuclear iransfer of a female pronucleus into a
zygote from which the male pronucleus has been removed, result in embryos which develop
relatively well yet lack proper development of trophoblastic tissues. Parthenogenetic embryos,
which differ from gynogenotes in that they are activated without fertilisation and retain the second
polar body, exhibit the same developmental features and failures as gynogenotes (Surani et al.,
1984)-
Conversely, androgenetic embryos (two paternal genomes) exhibit a very different
phenotype. The resulting embryos have hypertrophied trophoblastic tissues; however the embryo
is very poorly developed. These embryos die early in development (Surani et al., 1984; McGrath
and Solter, 1984). These studies have clearly illustrated that both the matemal and patemal
genomes are essential for proper development.
The focus of studies which followed these observations had the specific aim of trying to
identify which regions of the mouse genome are functionaüy nonequivalent in parental genomes.
This was accomplished by generating rnouse embryos which had uniparental disomies for specific
chromosomes or chromosomal regions through intercrossing mice with balanced translocations.
An imprint map (Figure 1) was successfully generated for the mouse genome identiQing eight
autosomal regions which exhibit differential effects depending on their parent of ongin (Cattmach
and Kirk, 1985). Six to seven of these regions are localized to chromosomes 2, 7, and 17. Two
other regions are on chromosomes 6 and I 1. Matemal and paternal disomies on chromosomes 1,
5,9, and 14 have also been recovered at different frequencies, which may result from the effects of
unknown imprioted genes.
The functional difierences between matemal and patemal genomes were clearly illustrated
Figure 1: Autosornai chromosome imprinting map of the mouse. Chromosomes are depicted to
show region of normal and defective complementation on (M) maternai and (P) paternd
homologs. (Taken from Cattanach and Beechey, 1990).
........... ..... dlfi erentlal recovery untested
6 in the above experiments. and experiments which followed to the present date have identified
specific genes which are subject to genomic imprinting. It has been estimated that approximately
100-200 imprinted genes may exist in the mouse genome (Barlow, 1995). The seventeen
imprinted genes identified thus far have been found by examining regions of the chromosome
which are associated with estabiished irnprinted regions (p57ki~2, Hatada and Mukai et ai.. LWS),
systernatic screens such as differential expression assays (PeglMest, Kaneko-Ishino et ai., 1995)
and restriction landmark genomic scanning with methylation sensitive enzymes (UZafb-n,
Hayashizaki et al., 1994), or by serendipity (Igf2, DeChiara et ai., 199 1; H19, Bartolomei et ai.,
199 1) Some correlations have been made about these irnprinted genes with respect to chromosome
location, expression pattern and biological function. Differences do exist between humans and
mice, where some imprinted genes in the mouse have ken evolutionarily conserved although their
imprint stanis has not been, such as the U2af binding protein related sequence 1 (UZAFBP-RS),
INS2 (Nakao and Sasaki, 1996) and IGF2R (Ogawa et al., 1993).
1.2.2 Models of Irnprinting
Irnprinted genes are initially characteriseci by their allele specific expression patterns. How
these alleles are silenced however has been the subject of much debate. What is clear about these
genes is that their irnprinting is dependent on complex regulation. The IGF2 (Insulin-like growth
factor 2) gene in the human liver changes from monoallelic expression in the fenis to biallelic
expression after birth (Nakao and Sasaki, 1996); the paternal allele of the mouse Igf2 gene is
expressed in both embryos and adults, however in the choroid plexus and leptomeninges of the
adult, biallelic expression is observed (DeChiara et al., 199 1); and the Igf2r gene in the mouse is
expressed from both alleles until later in post-implantation development, where its expression
becomes monoallelic (Szabo and Mann, 1995b). The mode by which these changes are achieved
is unknown. Various models have been proposed to explain how genomic imprinting occurs.
Results obtained by numerous investigators suggests that the "imprint" may be a gene specific
mark andor a chromosome domain phenomenon.
DNA methylation is one exarnple of an epigenetic modification which occurs in the nucleus
and may regulate the specific expression of certain genes (Monk, 1995). Transgenic studies in
mice have shown that some transgenes are heavily methylated when materndly inherited and
undermethylated when patemally inherited (Sapienza et al., 1987). Most imprinted genes studied
7 to date display differential methylation between parental alleles. However, in examining
rnethylation patters in garnetes and early embryos, apparent differences in methylation were not
observed until later stages of development. Both alleles of the Igf2 gene are methylated in the
oocyte and sperm and differences in rnethylation are not observed until the blastocyst stage
(Brandeis et al., 1993). In contrast, Hl9 has paternal specific methylation in a region upstream of
the promoter during spermatogenesis. IgEr also has a materna1 specific mark during oogenesis
supporting the idea that methylation rnay be the imprint mark. Experiments snidying the early
deveiopment of the embryo revealed that there is a loss of methylation between the eight ceII stage
and the blastocyst stage of development. It has been proposed that this imprint mark is resistant to
demethylation and remains unaltered during de novo methylation, in nirn, distinguishing between
the parental alleles (Barlow, 1993). It has k e n suggested that this massive dernethylation may
provide a mechanism of erasing the gametic imprinting which resulted from the parental genornes.
De novo methylation is then observed at the time of implantation. k i n g f i t detected in the inner
cell mass of the embryo. This de novo methylation also occurs independently in the
extraembryonic lineage and in the three different genn layers which allow for differences in the
patterns of methylation (Monk, 1995).
The role of DNA methylation in the maintenance of monoallelic expression of imprinted
genes in the mouse was established with the creation of a nul1 mutation in the DNA
methyltransferase (Dmnt) gene. Dmnt -1- mice died at &y 1 1 of embryonic development, and their
genomic DNA was substantially demethylated. HL9 gene expression was elevated, demonstrating
that DNA methylation is essential for maintaining the transcriptionally inactive state of the parental
Hl9 allele. Igf2 expression was also dependent on Dmnt for normal expression levels of this
gene. Igf2r however gave different results, in that RNA isolated in day 9.5 mutant embryos did
not differ in Igf2r expression when compared to wild type embryos. AIthough this gene was
demethylated, its expression was resistant to these changes suggesting that DNA methylation may
not be required for the maintenance of the Igf2r impnnt. The relaxation of imprinting in these
Dnmt deficient rnice provides evidence that methylation is required for the maintenance of
monoallelic gene expression for most irnpnnted genes (Li et al., 1993). ES cells, homozygous for
the Dmnt deletion are fully viable with their DNA mostly dernethylated. Upon differentiation,
these ES cells undergo rapid ce11 death suggesting that DNA methylation has an important role in
later stages of development (Panning and Jaenisch, 1996). This finding supports the results
8 obtained in the generation of a nul1 mutation of this gene in mice which die s w n after gastrulation
(Li et al., 1992). Current work by Tucker et al., (1996) have rescued the ES cell lethal phenotype
and restored the overail methylation level to normai by the introduction of a Dnmt minigene. The
Igf2r and HI9 deles were not remethylated at either allele and normal monodelic expression was
not restored unless the unmethylated aiieles passed through the germ iine.
If methylation were the m e imprint mark, it would be expected thai imprinted genes would
be monodlelically expressed from gametogenesis throughout development (if the mark was
resistant to demethylation) or at the blastocya stage (if a new mark were laid down). However this
does not occur suggesting that there must be some other modification which occurs during
development. RNA RSH could i d e n w the precise time point at which bialielic expression is
replaced by monoailelic expression in cells of various tissues.
In the mouse genome, imprinted genes appear to occur in clusters suggesting that
irnprinting may be a chromosome domain phenornenon. The mouse chromosome 7 contains two
separate regions where imprinted genes reside. The imprinting cluster on the distal end of this
mouse chromosome which includes Mashî, Igf2, Ins 2, H l 9 and p57kip2 is syntenic to a region
on human chromosome 11. A second cluster on rnouse chromosome 7 is syntenic to a region on
human chromosome 15 which includes Snrpn, Znf127, PARI, PARS and IPW. Another possible
cluster may exist on a region of the mouse chromosome 17 which contains Igf2r, Mas and fused, a
gene which experiences unequal penetrmce of the parental aileles but is not imprinted (Ruvinsky
and Agulnik, 1990), consistent with the studies of Cattanach and Kirk (1985). Altered regulation
of these imprinted domains is responsible for human genetic diseases. The Beckwith-Wiedemann
syndrome may result from a double dosage of the Igf2 gene in humans and defective Hl9 gene
expression. Deletions which encompass the Snrpn gene on the human chromosome 15q11- 13 are
linked with two disorders, the Prader-Willi syndrome (PWS) and the Angelrnann syndrome (AS)
(Nakao and Sasaki, 1996). Buiting et al., (1995) have shown that a small deletion at this locus can
impair the imprinting in this region and cause these two distinct genetic diseases. The deletion has
been proposed to contain an imprinting control centre where, under normal conditions, the AS
genes are expressed from the matemal allele and the PWS genes are expressed from the paterna1
allele only. Deletions which include the imprinting centre on the patemal chromosome silence the
expression of the PWS genes and allow for the expression of the AS genes. resulting in the PWS
phenotype. Similarly, a deletion on the matemal chromosome silences the AS g e n s and causes
9 the inappropriate expression of the PWS genes resulting in the AS phenotype.
The distal end of chromosome 7 in the mouse is the focus of extensive studies which
attempt to detemine the importance of these clustenngs. IgfZ and Hl9 are two imprinted genes
which are oppositely imprinted, that is the H 19 gene is piuemally silenced, and the maternai d e l e
is transcriptionally active, while for the Igfî gene, the paternal ailele is monoallelicdly expressed
and the matemal allele is silenced. Since these two genes are closely linked, they have been
studied in depth and have yielded the "ehancer cornpetition" mode1 which attempts to explain how
these genes are oppositely imprinted (Leighton et al., 1995). Expression of the Igf2 gene is
dependent on the interaction of the Hl9 gene with two enhaocen which lie downstream of the
gene. Recent experiments have also shown that if the active matemal allele of the H l 9 gene is
deleted, biallelic expression of the Igf2 gene is observed, providing more evidence that Hl9
expression is vital for the maintenance of the Igf2 imprinting (Ripoche et al., 1997). Examining
the interphase nucleus would illustrate this mode1 best by detecting the nascent transcnpts of each
gene and observing the exclusive monoallelic expression of Hl9 and Igf2 on opposite
chromosomes.
CeIlcycle replication time is one parameter that has been s h o w to be associated with tissue
specific gene expression (Kitsberg et ai., 1993). Imprinted genes have been shown to replicate
asynchronously, which contrast with the typical synchronous replication timing of non-imprinted
genes. The replication timing patterns for chromosome regions containing the Igf2, H19. Snrpn
and Igf2r genes were examined using DNA in situ hybridization to interphase nuclei. Results
have shown that the paternai allele is early replicating. This allele-specific replication timing
appears to be a special characteristic of imprinted gene regions. Later studies by Kawarne et al
(1995), have shown that some genes which exhibit replication asynchrony by FISH are in fact
replicating synchronously when assayed by a PCR based assay. The authors postulate that the
differences between parental alleles may reflect structural differences between the matemal and
paternd alleles rather than differences in replication timing, for example association of newly
replicated chromatids.
Nonequivaience between parental genomes has also been observed in mice with respect to
X chromosome inactivation. Although inactivation of one X chromosome in femaies is generally
random in humans and most mouse tissues, it has been shown that the patemal X is preferentially
inactivated in the patemal g e m line in the extraembryonic tissues of the fernale rnouse (Talcagi and
10 Sasaki, 1975). Studies on X inactivation proposed the existence of an imprinting centre on the
inactive X which may be responsible for the silencing of n&y genes. The imprinting centre on
the inactive X has been referred to as the X inactivation centre from which Xisr, a large X-inactive
specific transcxipt, which like H19, does not encode a functionai protein, is actively transcribed.
This transcnpt has been shown in humans (Clemson et al.. 1995) and in mice (Panning and
Jaenisch, 1996) to coat the inactive X chromosome in the nucleus. This centre is responsible for
the initiation and spread of X inactivation as well as its maintenance in a silenced state. X
chromosome inactivation has been shown to act in cis and to initiate from the single site of the X
inactivation centre, which has been localised to a 450 kb region of the mouse X chromosome
through transgenic studies (Lee et al., 1996). Integration of Yac transgenes carrying Xist into
dflerent regions of autosomal chromosomes displays some properties of the X inactivation centre
(Lee and Jaenisch, 1997). Ectopic Xist RNA completely coated the transgenic chromosome 12,
reducing the expression of this chromosome's genes two-fold In addition, ectopic X-inactivation
centres showed characteristic changes to the chromosome such as delayed DNA replication and
hypoacetylation of histone H4 (Lee and Jaenisch, 1997). It has recently been demonstrated that
high level Xist expression from the inactive X chromosome is regulated by the accumulation and
stabilization of the Xist transcripts a< the inactive X. rather than stimulation of Xis t transcription
(Panning et al., 1997). suggesting a post transcriptional mechanism for X inactivation.
1.2.3 Allele specific patterns of expression of imprinted genes
The time at which an imprinted allele is transcriptionally silenced was established in a series
of experiments undertaken by Szabo and Mann (1995b). They exarnined the allele specific
expression of four impnnted genes, IgfZ, Igflr, Hl9 and Snrpn, during normal preimplantation
and early postimplantation development of the mouse. The focus of this snidy was to determine at
what stage epigenetic modifications are in place to cause monoallelic expression patterns of these
four imprinted genes. If the epigenetic system was fully established by the time of genome
activation, imprinted genes would exhibit monoallelic expression by the late two cewearly four ce11
stage, which corresponds with the time of zygotic genome activation. However, if the expression
of the imprinted genes is biallelic at the time of genome activation then it would lend support to the
idea that there must be additional epigenetic events which occur after the 8 ce11 stage. This work
has nicely established the time at which the imprinted genes in this study undergo monoallelic
11 expression. Using the RT-PCR SNuPE andysis. it was shown that Snrpn, monoallelic expression
is fully established by the time of zygotic genome activation. With Igf2, the imprint status was
detennined prior to zygofic genome activation since it was monoallelically expressed in tissues up
to the 8 ce11 stage; however, biaelic expression was detected at the blastocyst stage. Studies by
Latham et al., (1994) and Mann et al., (1995) showed that Igf2 expression is biallelic in
parthenogenetic, gynogenetic, and androgenetic embryos. Biallelic expression is also found for
Hl9 in isolated 6.5 dpc tissues showing that this gene is not subject to imprinting repression at the
initial stage of high expression. Igf2r was highly expressed during preimplantation development
as was Snrpn; however its mode of expression was bidlelic. Levels of Igf2r RNA during
postimplantation development were lower than during preimplantation development, and
monoallelic expression was apparent in visceral endoderm but not in other tissues, illustrating its
tissue specificity. The processed mRNA collected in the study of Szabo and Mann resulted from
pooling all tissues and therefore is not capable of detecting monozllelic expression for individual
cells of a tissue.
Similar work was also done by these same authors to determine the expression of the sarne
imprinted genes in the mouse germ Iine. Their findings clearly illustrate that the expression of
these genes in primordial germ cells upon entering the embryonic genital ndge at 1 1.5 dpc and
throughout garnetogenesis are al1 biallelic. Hl9 was the only gene whose RNA could not be
detected in late gametogenesis. The results have suggested that the imprint status of these genes
from the parental genomes are erased. and that the new imprint status may not take place until late
in garnetogenesis. Therefore, either the imprint is erased and not reestablished in the germiine until
after day 1 1.5 of embryonic development, or expression is insensitive to the imprint in these cells
(Szabo and Mann, 1995a).
1.3 DYNAMICS OF THE NUCLEUS
The nucleus is a dynamic and cornplex structure and cytological studies have shown that
although there are no distinct membrane bound compartments, it is highly organized and contains
hinctional regions. Al1 of a cell's genetic information is contained within this minute space, and
depending on the tissue to which this nucleus belongs, its levels of transcription and expression are
highly variable, yet under strict control. The method by which this is achieved is the focus of
rnany shidies. Dunng the interphase stage of the ce11 cycle, the appropriate genes must be actively
12 transcribed at precise times, the pre-mRNA moledes must be processed and transported through
various areas of the nucleoplasm to localize either in the nucleus or to be transported out through
nuclear pores into the cytoplasm as &A. The areas in which the RNA processing occurs were
found to be highly ordered, and contain neighbouring snRNP ( smd nuclear ribonucleoprotein
particle) clusters which exist in a reticular network that extends between the nucleolar surface and
the nuclear envelope (Spector, 1990). It is at these s n R N P clusten that the pre-mRNA gets
processed that is, the introns get spliced out, and the snRNP reticulum provides pathways by
which the processed mRNA fmds its way to the nuctear pores.
1.3.1 The interphase nucleus
Most eukaryotic DNA is expressed and reproduced in the interphase nucleus. Chromatin
can be classified as heterochromatin and euchromatin based on its state of condensation during this
phase of the ceIl cycle. Heterochromatin is highly condensed during interphase and is therefore
considered to be transcriptiondly inactive. It is further divided into constitutive heterochromatin,
which is highly enriched in repetitive sequences such as alpha satellite sequences and comprise
approximately 10% of the genome, and facultative heterochromatin, which consists of potentially
active chromatin and involves the whole chromosome, or a pair of chromosomes, best exemplified
in X inactivation where one X chromosome in femde placental mamrnals is condensed into a Barr
Body (Spector, 1993). In the average eukaryotic cell, approximately 90% of the chromatin is
transcriptionally inactive at any given time, but al1 of the inactive chromatin may not be in a
condensed state. About 10% of the chromatin that is transcriptiondly active is always in the form
of decondensed euchromatin (Manuelidis, 1990).
1.3.2 Nuclear speckles
During or after the transcription of pre-&A, transcripts must be processed and then
transported to the cytoplasm where they are tmnslated into proteins. For most transcnpts, this
includes the addition of a 7-methyl-guanosine cap structure at the 5' end, heteronuclear
ribonucleoprotein (hnRNP) assembly, splicing of noncoding intron regions, subsequent ligation of
exons, polyadenylation, and the exchange of hnRNP proteins for mRNP proteins (Spector, 1993).
Many studies have determined what the spatial organization of the pre-mRNA is with respect to its
splicing components and the nuclear structure. Immunofluroescence microscopy has found that
13 snRNPs as well as several non-snRNP splicing factors (such as SC-35 which is a spliceosome
assembly factor) are localized in a speckled pattern throughout the nucleoplasm. Poly (A)+ RNA
has been shown to colocalize to 20-40 discrete nuclear speckles. Further examination at the
electron microscope level has revealed that these speckled patterns refer to the interchromath
granule clusters and perichrornatin fibrils. Interchromath granule clustea are the sites of splicing
factor storage and assembly and these splicing factors shunle between these clusters. The
perichromaîh fibrils are the sites of active transcription (Spector et al., 1993).
1.3.3 Nuclear tracks
Extensive studies into the biochemical steps involved in pre-&A transcription and
processing are well known and characterized. The subnuclear localization of these events is
currently king investigated. Higher level nuclear organization is suggested by the highly l m
and spatially compartmentaiized distribution of individual genes and their cognate RNAs
(Lawrence et al., 1988 and Lawrence et al., 1989). Studies using the lymphoma ce11 line
(Narnalwa) which contain two copies of the Epstein-Barr Virus genome closely integrated on
chromosome 1. revealed compelling evidence on the organization of the nucleus. Ruorescent
detection using nonisotopic in situ hybridization has shown severai hundred copies of specific
RNA sequences (nascent transcripts) are tightiy constricted to a small region of the nucleus, in a
curvilinear track. Al1 of the specific RNA was detected to be confined within the distinct
boundaries of the track of focus, and they do not extend out or exhibit a difise distribution
throughout the nucleoplasm. These signals also appear to be larger than the typical DNA signals
visualized by DNA FISH (Lawrence et al., 1989). These results confirm that these RNA
transcnpts are precisely localized in the nucleus and argue strongly against the free diffusion of the
RNA, exempliQing that the nucleus is highiy stmctured and compartmentalized. The tracks also
provide evidence that they are transported directly to the nuclear membrane where the processed
RNA can be transported out of the nucleus via the nuclear pores.
1.4 RATIONALE
The time at which an imprinted gene is monoallelically expressed, or one of its alleles is
transcriptionally silenced is difficult to ascertain in individual cells. Typicaily expression patterns
of imprinted genes have been assessed by examining the steady state Ievels of mRNA in various
14 whole tissues as depicted in the snidies of Szabo and Mann (1995ab). 1t is unknown if a i l ceils of
a tissue respond to the epigenetic modifications which result in o d y one active allele. Probing the
interphase nucleus will permit the direct visualization of exactly what is occurring in each celI at the
time of fixation. Are both aileles k i n g actively transcnbed in a major@ of celis? 1s expression
le*, in that one allele expresses the transcript at a very high level. while the other is actively
transcribing the gene but at a much lower rate? And most importantly do all cells of a tissue act in
the sarne way.
RNA FISH possesses the ability to detect nanscripts near their site of synthesis and can
provide insight into the developmental regdation of gene expression directly in individual ceUs. It
can also be an accurate indicator of very recent transcription in contrast with other in situ
hybridization techniques which detect the presence of accumulating mRNA but cannot distinguish
ongoing or recent initiation of gene activity.
1.5 OBJECTIVES
1. 1 planned to optimize conditions of hybridization for the detection of biallelic expression of
fibronectin. the positive control gene, on a cultwed fibroblast ce11 line using RNA FISH.
2. After optirnizing conditions for RNA FISH on a cultured cell line. I attempted to modify the
technique for the detection of monoallelic expression patterns on isolated nuclei of 7.5 and 8.5 dpc
mouse embryos utilizing the H 19 and Igf2 genomic sequences as probes.
3. Utilizing this sarne technique. 1 attempted to optirnize the conditions for the detection of
monoallelic expression patterns of two irnpnnted gens. Hl9 and IgE?. in tissue culhire cells, and
blastocyst and ectoplacental cone outgrowths since it has k e n shown that the cells which constitute
the outgrowths abundantly express both Igf2 and Hl9 mRNA. Culturing of these cells allowed
me to exploit the expression of these irnpnnted genes in cells grown I virro.
CHAPTER 2
MATERIALS AND METHODS
2.1.1 Animals
Mice utilized for all experiments were of the CD-1 shah. These mice were chosen because
they breed well and large litter sizes can be retcieved per fernale after rnating.
2.1.2 DNA utilized as probes
Both cDNA sequences and genornic sequences were utilized as probes for RNA FISH.
Beta-actin cDNA in the pBR322 plasmid was propagated by a large plasmid prep as described by
Sambrook et al (1989). The size of the cDNA is 1.8 kb in a 4.4 kb plasmid. The genomic
sequence of Hl9 in the pUC9 plasrnid was also propagated as above. H 19 is 3.0 kb, including ail
its introns and exons. The total size of both plasmid and insert is 5.7 kb. Al1 probes were left in
the plasmid for the nick translation reaction since it has k e n shown by Lawrence and Singer
(1985) that the plasmid participates in network formation to provide an increased hybriditation
signal. The Igf2 mouse genomic sequence, which is approximately 19 kb, was obtained from
Marisa Bartolomei (Howard Hughes Institute, Philadelphia). The rat fibronectin cDNA was
utilized as the control probe and obtained from Barbara Panning (Whitehead Institute. Cambridge).
The cDNA sequence of this gene is approximately 20 kb.
2.1.3 Solutions, Glassware and Plasticware
AU solutions listed and prepared for this work were RNAase free. To minunize the activity
of RNAases, either endogenous or exogenous, solutions were made in diethyl pyrocarbonate
(DEPC) (Sigma) treated water. DEPC was added to Milli-Q deionized water at a concentration of
0.1% and placed in a shaker ovemight. The following moming, the water was autoclaved for
twenty minutes. A11 solutions were prepared using RNAase-free glassware, and DEPC treated-
water. Sterile, disposable plasticware was extensively used since they are essentiaily fiee from any
RNAases. Ail glassware, coverslips, slides and coplin jars were baked a& 550 C for at least four
hours to destroy any RNAases which may exist (Sambrook et al., 1989). Ail slides were
15
16 precleaned with 70% ethanol prior to baking. Unless otherwise stated, RNAase-free conditions
were used for ail experiments.
2.2 METHODS
2.2.1 RETRIEVAL OF MOUSE EMBRYOS
CD4 females were caged with CD4 males ovemight and checked for vaginal plugs the
following morning. The day of the plug was counted as king &y 0.5 of development. Females
were removed and monitored until the proper day of development for the retrieval of embryos.
Days of interest for embryonic development were typicdy days 3.5,7.5, 8.5, and 10.5.
2.2.1.1 Superovulation of females for retrieval of mouse blastocysts
In order to snidy tissues which give rise to extraembryonic cells and evennially the
placenta, female CD4 mice were superovulated by injection of 5 IU of pregnant mare serum
(PMS) (Sigma) subcutaneously, followed by an injection of 5 IU of human chorionic
gonadotropin (HCG) (Sigma) 48 hours later. Females were then caged with males overnight and
checked for vaginal plugs the following moming. Females were sacrificed at day 3.5 of
embryonic development and blastocysts were flushed from the genital tract into KSOM medium
and cultuted as descnbed below.
2.2.1.2 Dissection of post implantation embryos
At the appropriate stages of embryonic development, fernales were sacrificed and dissected
for the retrievai of embryos. The decidua were collecteci in Phosphate Buffered Saline (PBS) (137
mM NaCI; 2.68 rnM KCI; 10 rnM Na2KPO4; 1-76 m M KH2PO4; pH 7.4), and embryos were
recovered. Reichert's membrane was removed and embryos were grouped as follows: day 7.5
embryos were dissected into three parts; the ectoplacental cone, extraembryonic ectoderm and
embxyonic ectoderm; day 8.5 embryos were dissected into the placenta and embryo; day 10.5
embry os were dissected and cul tured ovemight as descnbed below (see Section 2.2.2.2).
2.2.1.3 Retrievai of nuclei
To isolate interphase nuclei for RNA FISH, the tissues which were dissected were pooled
17 and placed into a microfuge tube. Tissues were dissagregated by trypsinization using 0.5 m g M
trypsin and 0.53 mM EDTNPBS (Gibco) at 37 C. Treatment with trypsin was carried out for
various times as shown in Table 3. Longer trypsinization times, from 30 minutes to 75 minutes,
were f i t employed for tissues of the ectoplacental cone and placenta since it appeared that these
cells were more difficult to dissociate. However, trypsinization times were later reduced to 1-2
minutes to reduce RNA degradation. Trypsin was inactivated by the addition of 10% fetal bovine
serum for 10 minutes at room temperature. Ceils were then peileted at 189 g.
Cells were further washed with lxPBS to remove any traces of senun and trypsin, and
pelleted again. The ce11 pellet was resuspended in a cold cytoskeietal buffer consisting of 100 m M
NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM PIPES pH 6.8 and 10 m . vanadyl ribonucleoside
complex (VRC) and incubated for 30 seconds. Cells were pelleted, and resuspended in
cytoskeletal buffer plus 0.5% Triton X-100 (Sigma) for various times (Tables 2 and 3). Triton X-
100 was included to remove the cytoplasm component of the cells and leave behind intact nuclei.
Nuclei were then spun down and washed with the cold cytoskeletai buffer without Triton X-100
for 30 seconds. Nuclei were pelleted again and then fixed at room temperature with 4%
parafonnaldehyde in PBS, pH 7.5 for 10 minutes. Nuclei were applied to polarised (positively
charged) slides (Fisher) within a space on the slide encircled by the tracing of a diamond pencil,
and left to air dry at room temperature for a minimum of 30 minutes.
2.2.1.4 Storage of nuciei
After nuclei were irreversibly bound to the polarised slides, slides were washed with 70%
ethanol and stored at 4C in 70% ethanol for up to two months. Longer times in 70% ethanol have
been reported to improve detection of RNA by FISH (Lawrence et al., 1989).
In order to assess whether the dissection and treatment of the nuclei was successful and did
not result in any morphological damage, at least one slide from each of the components of the
tissues was stained with DAPI to verify the yield of nuclei. It was very important to get cells in a
monolayer for FISH. If the DAPI staining displayed damaged, or unevenly dispersed nuclei, the
slides were discarded.
2.2.2 TISSUE CULTURE
2.2.2-1 Mouse fibroblast ce11 line
A mouse embryonic fibroblast ce11 line was cultured to yield monolayers of cells.
Fibroblasts were plated in 90mm diameter petri dishes in Dulbecco's Modifed Eagle Media (High
Glucose) suppiemented with ImM sodium pymvate, 0.1 mM non-essential amino acids, 55 uM
$-mercaptoethanol, 2 mM L-glutamine, penicilIin/streptomycin, 10% fetal bovine serum, and
p w n in a humidified 37 C incubator in presence of 95% air, 5% C02. When ceUs reached 100%
confluence, they were typsinized for approximately 5-10 minutes at 37 C. Cells were resuspended
in culhue media, and dispersed into two dishes cootaining gelatin coated coverslips. The 18mm x
I8mm coverslips were prepared by baking to remove RNAase. and then coated with 0.1% gelatin
to provide a sticky surface to which the fibroblasts could adhere. As the cells grew to 100%
confluence, they were permanently aîtached to the covenlips.
At coofluency, the media in the culture dishes was removed and covenlips were washed
extensively with PBS. Coverslips were then exposed to the sarne cold cytoskeletal buffers as
described above, for varying times (Tables 1 and 2). Coverslips. with cells anached, were fixed
with 4% paraformaldehydeîPBS for 10 minutes and washed with 70% ethanol. Some coverslips
were also fixed with 4% pdormaldehyde/PBS/ 0.1% cetlypyridinium chloride (CPC) (Sigma).
Covenlips were left in the petri dishes and dishes were topped with 70% ethanol for storage at 4
2.2.2.2 Culturing of post implantation embryos
Embryonic cells were cultured overnight following tissue dissociation to provide the cells
with an opportunity to recover from dissection and to attach to coverslips in a monolayer
facilitating their use for RSH. Following dissection, tissues were pooled from approximately 12
embryos and dissociated by warm trypsinization as described above. Following trypsin
inactivation and washes with PBS, cells were seeded on O. 1 % gelatin coated coverslips and topped
with culture media Petri dishes were incubated ovemight and cells fixed the following &y as
described above. Cells on coverslips were also stained with DAPI to check the recovery of cells.
19 2.2.2.3 Culhiring of blastocysts and ectoplacentat cones for outgrowths
Mouse blastocysts were Bushed from femaies at day 3.5 of development and placed into
Lab Tek chamber slides (NUNC) topped up with tissue culture media. Slides were left in a 37 C
tissue culture incubator for approximately 4-6 days which was the length of time necessary for the
embryos to hatch from the zona pellucida and attach to the plastic surface. Primary trophoblast
giant cells flattened and spread out while the inner ceU mass celIs grew in a smaiI clump (Vannuza
et al.. 1988). After 6 days in culture, slides were washed extensively with PBS to remove any
culture media, and then fixed with 4% parafoITnaldehyde/PBS for 10 minutes at m m temperature.
Some outgrowths were treated with 0.5% Triton X-100 for 2 minutes before fixation since this
yielded positive resuits for the fibrcblast ce11 line. Outgrowths were hirther washed with 70%
ethanol to remove any remaining paraformaldehyde and stored in 70% ethanol at 4 C.
The same procedure was undertaken for ectoplacental cones of day 7.5 embryos. Embryos
were dissected free of maternai decidua and the ectoplacental cones were retrieved and placed into
chamber slides with tissue culture media. The slides were kept in a 37 C incubator for
approximately 4 days. Once the secondary trophoblast giant cells attached. they spread out and
grew rapidly. At this point, they were fixed as described for the blastocysts. However the Triton
X- 100 step was eliminated in these outgrowths.
2.2.2.4 Embryo Squashes
In order to concentrate a large number of cells within a confined area on a slide and at the
sarne time ensure that these cells are in a monolayer, embryo squashes were prepared. Embryos
were dissected out at 8.5 dpc but were iefi intact. Embryos were treated with the cytoskeletal
buffers and fixed with either 4% paraformaidehydelPBS, or 4% paraformaldehydelPBS/O. 1%
CPC. In some cases the treatment with cytoskeletal buffers was omitted. After fixation, a
coverslip was added to squash the embryo. The slide was then placed in an absolute ethanoVdry
ice bath for approximately 5 minutes. The covealip, being thimer than the slide. warmed to room
temperature quicker than the slide after removal from the ethanol. The covenlip was quicldy
removed leaving the squashed embryo attached to the polarized slide. The slide was submerged
once again into the ethanol dry ice liquid for 5-8 minutes and left to air dry. Slides were then
stored at 4 C in 70% ethanol.
Squashed embryos were processed sirnilarly to fibroblast cells in that ceils were treated
20 with 0.5% Triton X-100 in cold cytoskeletai buffers for two minutes prior to fixation. Other
squashed embryos were fured immediately, and prior to hybridization with the Igf2 genomic
probe, cells were treated with 0.5% Triton X-LOO in 0.2 N HCI.
2.2.3. FISH PROBES
2.2.3.1 Nick translation of probes
The Bio-Nick kit (Gibco-BRL) was ualized to generate biotinylated probes containing the
biotinylated-adenosine dinucleotide. Incorporation of the biotin- 16 dATP into the DNA sequence
of interest resulted from the nick translation reaction. One microgram of DNA was labelled as
descnbed by the manufacturers. 10 ul of the reaction mix was run on a 2% agarose gel to venQ
the fragment length of the nicking reaction (Figure 2a). Ideally, probes should range from 150-
300 bp in sue to yield optimal results in terms of signai intensity and background (Lawrence and
Singer, 1985). To test the incorporation of biotin in the DNA of interest the agarose gels were
blotted using the rnethod of Sambrook et al (1989). The nylon membrane was UV crosslinked and
biotin incorporation was visualized using the Detek-Hrp kit (Enzo Biochemicals) (Figure 2b).
2.2.3.2 Probe preparation
If the incorporation of biotin was successful. the bio-nicked fragments were prepared for
FISH. To remove unincorporated dinucleotides, the probe was precipitated with two volumes of
absolute ethanol, one tenth volume of sodium acetate (3M) and 10 ul of a 20 mg/ml of tRNA for
every 200 ng of probe. The probe was placed at -70 C for 30 minutes, centnfuged for 15 minutes
and resuspended in water. The probe was precipitated once again with the addition of 10 ul of
human Cot-1 DNA (1 mg/ml) (Gibco-BRL) and 20 ul of 10 mg/rnl of sonicated salmon sperm
DNA. Mer 30 minutes at -70 C, the probe was once again centrifuged for 20 minutes, washed
with 70% ethanol, 100% ethanol and dried. The pellet was resuspended in ultrapure deioinzed
formamide (Gibco-BRL). The probe was vortexed and pipetted to ensure al1 of the pellet was
resuspended. The probe was then placed at 37 C for a minimum of one hour to allow the Cot
DNA (unlabelled cornpetitor DNA), to bind with repetitive signais of the probe DNA and block any
excessive background signals which could result with the overnight hybridization.
Figure 2: Verification of (a) probe fragment size and (b) biotin incorporation. (a) Foilowing the
nick translation reaction, 10 ul of the DNA was run on a high percent agarose gel. Typically,
fragment lengths were 150-300 bp (lane 2) when compared to the standard (lane 1). (b) Detection
of biotin incorporation was verified with the Detek-Hrp kit. If samples were not run on a gel, then
dot blots were made to test the incorporation of the biotin molecule in the DNA.
2.2.4.1 Dehydration of Slides
Slides a d o r coverslips treated prior to fixation with 0.5 % Triton X-100 were usually
dehydrated through an ethanol series of 70%.8O%. 95% and 100% for 2 minutes each pnor to the
addition of the probe for hybridization. Siides were Ieft to air dry.
Slides and/or coverslips which were treated with Triton X-100 or Proteinase K after
fixation were rehydrated initiaDy to prepare the nuclei for treatments with the enzyme or detergent.
Rehydration consisted of washing siides through an ethanol series of 70%. 50 %, and 30% for
two minutes each and then washing with PBS. Following various treatments with proteinase K or
Triton X-100 (Tables 1 and 2) slides were washed with PBS and then dehydrated through an
ethanol series of 70%. 808,95% and 100% and left to air dry.
2.2.4.2 Application of prepared probes
Following the one hour incubation at 37 C, the probe was denatured at 70-85 C for 5
minutes. The probe was placed on ice and approximately 20 ui of the probe mix was mixed with
20 ul of the 2x hybridization mix which consisted of 1 part 20xSSC, 1 part 2mM VRC complex, 2
parts of 50% dextran sulphate and 1 part of bovine serum albumin (10 mg/ml) (New England
Biolabs). Probe and hybridization buffer were vortexed briefly, applied onto the slide and covered
with a 22mrn x 40mm baked glass coverslip. The coverslip was sealed onto the slide using rubber
cernent. If cells were grown directly ont0 the coverslips, then the probe was added to the coverslip
and the coverslip was then inverted ont0 a clean baked slide and sealed with rubber cernent. The
slides were then placed into a humid chamber at 37 C overnight. The humid chamber consisted of
a large stede petri dish with Whatmann paper lining the battom and water to keep the chamber
hurnid. The slides were raised above the paper using autoclaved toothpicks. The humid chamber
was placed at 37 C approximately 4 houn pnor to use in order to ensure the chamber was humid
and at the proper temperature before the slides were placed inside for the ovemight hybridization.
Controls were included with every experirnent. After dehydration of the slides. the nuclei
were treated with RNAase A ( 10 uglml) in a 0.1 M Trido. 15 M NaCl solution, or 5 ul of RNAase
A was added directly to the probe hybndization mixture. At 37 C overnight, any endogenous
RNA would be degraded and result in no hybridization. Conml slides were hybndized ovemight
24 in a separate chamber to ensure no RNAase contamination wodd spread to the experimental slides.
2.2.5. DETECTION OF NASCENT TRANSCRIPTS
2.2.5.1 Removal of excess non-specïfic binding of probe
Following overnight hybridization, the nibber cernent was peeled off the slides with
forceps and slides were washed in coplin jars. Slides treated with the RNAase were washed
separately. Washes included three 5 minute washes, each at 39 C in a shaking water bath, with
50% deionized formamidel 2xSSC in order to remove any nonspecific binding of probe to non
target sequences. Three five minute washes of 2 x SSC at 39 C followed. One 10 minute wash of
1 x SSC and one five minute wash of 4 x SSC at room temperature concluded the extensive
washes to remove any unbound probe. At no point during any of the washes were the slides
aiiowed to air dry.
2.2-5.2 Blocking and Detection of Probe Location with Fluorochrome
To ensure that only the fluorochrome conjugated to avididstreptavidin bound to the
biotinylated hybnd cornplex, a blocking step was incorporated. Slides were incubated at 37 C for
20 minutes in blocking buffer which consisted of 4 x SSC and 4 m g h l of BSA, and then in a
detection buffer which consisted of 4xSSC and 1 mg/& of BSA with a 1:200 dilution of the
appropnate fluorochrorne for 40 minutes at 37 C. Two fluorochrornes were used: FïïC-avidin or
Texas red-avidin.
2.2.5.3 Counterstaining and Viewing of Slides
Following incubation in detection buffer, slides were washed with 4xSSC for 5 minutes at
room temperature in the dark, with shaking. The next step included 4 x SSC, 0.1% Tween-20,
and 1 u g / d of DAPI or 10 ug/ml of ethidium brornide for 5 minutes. Slides were finally washed
with 4 x SSC to remove any excess countentain. Slides were mounted with propyl gallate, the
antifade solution for the fluorochrome or 50% glycerol and covered with a coverslip. The
coverslip was sealed with nail polish and stored at -20 C until viewed by a Zeiss Confocal Laser
Scanning Microscope (Version 3.80). Ail specimens were viewed using the 63x oil immersion
objective.
CEAPTER 3
RESULTS
3.1 Embryonic fibroblast cell line
Fibronectin gene expression was utilized as the control in this thesis to optimize the
conditions of hybridization for the RNA FISH protocol. Fibroblasts were grown directly on
gelatin coated coverslips until they reached 100% confluence. Since they were grown directly on
coverslips they did not require a trypsinization step pnor to fmaîion. Fibroblasts exhibit contact
inhibition as they grow in tissue culture. As a result cells extend and grow, remainuig very flat and
on average having a nuclear thickness of about IO microns. Once cells reached confluence a
number of different experiments were performed using fibronectin cDNA as a probe (Table 2) and
some results are shown in Figure 3. The RNA FISH technique was successful in detecting
fibronectin nascent transcripts on the fibroblast cell line. Nascent transcripts were easily localized
using this technique illustrating the importance of utilizing monolayers of cells which abundantly
express the gene of interest. On average 56% of ai l observed nuclei had RNA FISH signals in
contrast to L .3% of RNAase treated control fibroblasts (p<0.01, chi-square test)(Figure 4).
A series of experiments was aiso undertaken to test various treatment regimes (Table 2;
Figures 5 and 6). Using fibronectin as a probe, results indicate that if cells were fixed fint, the
nuclear signals were undetectable. Figure 5 iilustrates that a gentle treatment with 0.5% triton-X or
5 u g M of proteinase K (after cells have been fixed with paraformaidehyde) does not allow the
probe access into the nucleus and the nascent uanscripts could not be detected. Extended
treatments with 0.5% Triton X-100 were performed to see if this could allow the probe to
penetrate, with tirnes ranging from 10-30 minutes (Figures 6-7 and 8). Prolonged treatment with
0.5% Triton X-100 for 30 minutes after fixation with 4 1 paraformaldehydelPBS only reveded
that 3.9 % of the observed nuclei had one positive signal (Figures 6e and f) (results are not
significant, ~ 4 . 0 5 ; chi-square test). None of the nuclei showed two û;rnscnptionally active alleles
as seen when fibroblasts were treated with 0.5% triton pnor to fixation (see above). If CPC was
included in the fixative then no signals were observed even after 30 minutes. Treatments longer
than 30 minutes were not preformed since such treatments were too harsh for the nuclei of the
dissected embryos. No differences were noted between the two methods of fixation, that is
supplementing the paraformalde hyde w ith CPC (Figure 6).
25
Figure 3: Treatment of nuclei with 0.5% Triton X-100 @or to fixation revealed the two actively
transcribing deles of the fibronectin gene (arrows). (a and b) 2 minute treatment. The mRNA is
aiso detected; (c) 5 minute treatment; (d) Conml nucleus with no positive signals. This image was
overexposed to show no transcripts could be detected Magnification -2300~.
c. - E 2 YI. Y
e P. C .- E V)
........
Figure 5: Fibroblasts were treated after futation for 5 minutes. (a) A O.S%Triton X-100
treatment was perfomed to see if any transcripts couid be detected Only mRNA could be detected
in aii cells examined; (b) Control ceHs were also treated with Triton X-100 and RNAase A; (c) 5
ug/ml of proteinase K was utilized, however only cytoplasmic staining was observed; (d) Control
ceiIs treated with the same concentration of proteinase K and RNAase A. No mRNA was detected.
Magnification - 1000~- 1 800x.
Figure 6: Only fibronectin mRNA couid be detected if the 0.5% Triton X-100 treatment was
performed after the fixation step. (a and b) 10 minutes; (c and d) 20 minutes; (e,f,and g) 30
minutes. In (e and f ) one signai (arrow) couid be detected but only in a srnail population of nuclei,
in cornpuison to (g) where only the rnRNA is observed (arrow). (a,c,e,f) were fixed with 4%
paraformddehyde/l x PBS, while @,d,g) were fixed in 4% paraformaldehyde/l x PBSl0.196
CPC; (h) Control fibroblast treated for 30 minutes with Triton X-100 and RNAase A prior to
hybridization. rnWA couid not be detected (arrow). Magnification -2000~-2300~.
37 Since the fibronectin transcripts could be detected, various probes were tried on the
fibroblast cells, such as the H l 9 and IgfZ genomic sequences. Fibroblasts were treated with the
identical conditions that were used to detect the biallelic expression of fibronectin. This included
treating the fibroblasts with 0.5% Triton X-100 for two minutes prior to fixation. For both of
these genes, no transcripts, either nascent or mRNA, codd be detected, suggesting that Hl9 and
Igf2 are not transcribed at levels in fibroblast cultured cells abundant enough to be detected by
FISH.
3.2 Analysis of day 7.5 embryonic nuclei utifizing RNA FISH.
In order to observe the nascent transcripts of various genes in embryonic nuclei, day 7.5
embryos were examined. At this developmental stage it is known that both Igf2 and Hl9 are
monoallelically expressed. RNA RSH should reveal hernizygous expression of Igf2 and H19,
and bialleiic expression of the control gene, B-actin, in various tissues of the embryo. Following
the dissection of embryos at the proper developmentd stage, cells were dissociated by warm
trypsinization. Cells were then exposed to cold cytoskeletal buffers prior to fixation. These
cytoskeletal buffers contain a Vanadyl Eübonucleoside Complex (VRC) which is a ribonuclease
inhibitor used during ce11 fractionation and the preparation of RNA. It does not allow for the
leaking of nuclear RNA and maintains the integrity of functional RNA molecules. This b a e r also
included the Triton X-100 detergent which acts to penetrate the nuclear membrane. Therefore
treatment of the nuclei with Triton prior to fixation allow for greater probe penetration and
accessibility to the nucleus. It has also been shown that longer treatment with Triton X- 100 allows
for better probe penetration, as well as aliowing for greater removal of the cytoplasm, leaving the
nuclei isolated and morphologically intact (Clernson et al., 1996).
Beta actin was the first probe utilized in these experiments since it is a housekeeping gene
and is highly expressed in al1 tissue types. The cDNA sequence of this gene was used. Attempts
to isolate the genomic clone of this gene were unsuccessful because the acth gene family is quite
large and possesses many pseudogenes (Minty et al., 1983). Actin RNA localizes inside the
nucleus and should be detectable with RNA FISH ( Xing et al., 1995). The mouse genornic H 19
gene was also used since it is known that Hl9 mRNA is monoaIIelically expressed at high levels at
these developmentai stages (Bartolomei et al., 1991).
Biotin-labelled probes representing specific fragments of the beta-actin cDNA and the Hl9
38 gene were hybridized ovemïght to these embryonic nuclei. Detection with texas red-avidin
revealed that parts of the nuclear membrane were labelled with this probe. However, no intemal
nuclear signals. observed by scannhg through the nucleus with optical sections of 0.5 microns,
could be detected which would represent the nascent transcripts of this gene. It appeared that the
probe could not penetrate the nuclear membrane and was bindlng to the nuclear envelope. It may
be specifically binding to the nuclear pores by which the processed B-actin mRNA exits the
nucleus. This suggests that the probe is not hybridizing to the pre-mRNA which is king actively
transcribed inside the nucleus (Figure 9). Prolonghg the 0.5% Triton X-100 treatment up to 10
minutes did not yield different results. Embryonic and extraembryonic cells produced the same
results.
3.3 Increasing accessability into the nucleus
Experiments similar to those described above repeatedly showed that the nuclear membrane
could not be penetrated, since signals accumulated at the nuclear membrane. Increasing the
accessability through the nuclear membrane became the focus of the experiments which followed.
Methods included increasing the length of time of Triton X-100 detergent treatment prior to
fixation, the treatment of nuclei with proteinase K prior to hybridization, and treatrnent with 0.5%
Triton X-100 in 0.2 N hydrochloric acid (HCl), followed by proteinase K treatment for various
times and concentrations prior to hybridization (see Table 3). As can be seen in Figure 10, the
treatments appeared to affect the nuclear morphology.
A select nurnber of experiments are described here in pa te r detail and are represented with
other experiments in Table 3. Day 7.5 and day 8.5 embryos were dissected and grouped into their
specific parts. The length of rime in which the cells were exposed to trypsin ranged from 30-70
minutes, as described above, since this treatment appeared to dissociate the cells appropriately.
Cells were treated with cold cytoskeletai buffers, varying the length of the Triton X- 100 treatmnt
from 30 seconds to IO minutes.
Treatment of &y 7.5 extraembryonic nuclei with proteinase K in the proteinase K buffer
for five minutes resulted in the destruction of nuclear morphology. Of 340 nuclei examined, only
three nuclei exhibited one signal as shown in Figure 10a; however this signal most likely does not
Figure 9 : Nuclei isolated from their cytoplasmic components after exposure to 0.5% Triton X-
100 for 5 minutes and hybridized with the bactin cDNA or H 19 probe. (a) pactin probe
accumulates on the nuclear membrane of extraembryonic cells. Signals are not intemal and do not
appear ubiquitously on the membrane (arrows); @) Only one nucleus from a group of ecto-
placental nuclei exhibit a nuclear membrane signal (arrow)with the beta actin probe; (c) Probe
accumulation was more pronounced on extmernbryonic nuclei hybridized with Hl9 (arrows); (d)
Control nucleus treaîed with RNAase A resulted in no probe accumulation anywhere on the
membrane. Magnification -2300~.
VI' VI 2. z: -. - sj 3, y y'
42
represent the nascent transcripts since $-actin has two active alleles. These results are not
statistically signifcant using the chi-square test (Pc0.05) when comparing the number of nuclei
with and without signals versus the control nuclei aeated with RNAase A.
Treatment of the embryonic ectoderm celIs with Triton X-100-HC1 and proteinase K (5
minutes with 30 ug/rnl) resulted in nuclei with intact morphology (Figure lob). However the large
nuclear signal was observed in only one per cent of al1 nuclei examined Day 8.5 embryonic cells
(Figure 10c) were treated only with 10 uglml of proteinase K for 5 minutes to reduce the
destructive effect of the proteinase K. The morphology was affected and the nuclei exhibited only
background staining. Gentle treatments with proteinase K or Triton X- 100 and hydrochloric acid
d e r fixing the nuclei with paraformaldehyde rarely gave positive results (< 1 %). In addition the
binding of probe to the nuclear membrane was abolished suggesting that the nuclear membrane
proteins were being digested and removed as a result of the various treatments. AU experiments
which used varying concentrations of proteinase K had the accompanying control slide where the
nuclei were treated with the same concentrations of the enzyme but also included the RNAase
treatment before the ovemight hybridization. The similarity of experirnental and control results
suggests that there may have been a problem with RNA degradation in the experimentd samples.
The source of RNA degradation was Iater discovered to result from the trypsin treatrnent which
occu~ed at the beginning of each treatment (B. Panning, peaond communication).
3.4 Mouse embryonic tissues
Various embryonic ce11 types were processed for RNA FISH using protocols established
in fibroblasts for the fibronectin probe. Cells used include 8.5 dpc embryos manually dissociated,
day 10.5 embryos also manually dissociated and cultured as monolayers in vitro, blastocyst
outgrowths, ectoplacentai cone outgrowths. and day 8.5 ernbryos squashed directly onto slides.
3.4.1 Embryos dissociated free of trypsin
8.5 dpc embryos were dissected and manually dissociated since it was suspected that
trypsin may be the source of contaminating RNAases. After dissection, tissues were teased apart
with forceps and vortexed to isolate individual cells. After neamient with the cold cytoskeletal
Figure 10: Increasing accessibility into the nucleus was tested by proteinase K treatments. (a)
Day 7.5 extraembryonic nuclei (stained with ethidium bromide) were treated with 20 ug/ml of
proteinase K for 5 minutes, and hybridized with the Bactin probe ovemight Only one weak
signal (mow), instead of two, is evident with the FITC-avidin fluorochrome (arrow); (b) Very
few embryonic ectoderm cells with the incorporation of the triton X-100 treatment in HCI
appeared to have a large nuclear signal (arrow) as detected with FTïC; (c) More gentle heatments
with proteinase K (10 ug/ml) of day 8.5 embryonic ceiis resuited in odd shaped nuclei which
exhibited only background levels of staining (arrow); (d) A control nucleus treated with 10 ughl
of proteinase K and RNAase showing no distinct signals above background. Magnification
- ~ ~ O O X - ~ ~ O O X .
45 buffers and fixation, nuclei were placed on polarised slides and allowed to air dry for
approximately 30 minutes. Ovemight hybridization with the Igf2 probe resulted in nuclei with
much of the probe bound to the nuclear membrane (Figure 1 la,b). Internai signals, investigated
by senal optical sections through the nuclei, revealed that no nascent pre-mRNA transcripts could
be detected at significant levels.
Culhired day 10.5 embryos were utilized for RNA FISH in parallel experiments with the
fibroblast ceus. The probe utilized for these experiments was the genomic sequence of Igf2. Afkr
embryos were dissected, these cells were also dissociated manually. Cells were isolated after the
washes with cold cytoskeletal buffers and peileted. For culturing, cells were resuspended in tissue
culture media, placed on gelatin coated coverslips and incubated ovemight at 37 C for up to 18
hours. One recumng problem was that cells did not remain attached to the gelatin coated
coverslips. Therefore, after the extensive washes involved in the RNA FISH procedure, a large
number of cells had been lost Where cells could be observed it was found that although the
treatments were identical to those used for the fibroblasts, no signais could be observed on the
cultured cells which differed significantly from the controls (Figure 1 1 c.d). It appeared as though
some probe always bound to the nuclear membrane regardless of treatment with the RNAase.
However, the nuclear morphology of these cultured cells appeared to be damaged.
3.4.2 Blastocyst outgrowths and ectoplacental outgrowths
Blastocyst and ectoplacental cone outgrowths were examined and treated for RNA FISH
using the Igf2 probe. Two treatments were utilized for fixing the outgrowths. Most slides were
fixed only with 4% parafomaldehydePBS. Some slides were treated with 0.5% triton prior to
fixation; however this damaged the outgrowths and they could not be studied. Most nuclei were
not in contact with the slides and since they grew in three dimensions. once the cytoplasrnic
support was removed it appeared that the nuclei were los& as examined by DAPI s a n g . A better
yield of cells was found when the outgrowths were fixed immediately. For each blastocyst
outgrowth, an average of 27 nuclei could be observed. Figure 12 shows a blastocyst and the
extent of its outgrowth after hatching from the zona pcllucida. Most of the cells which constitute
the outgrowth are pnmary giant cells which are known to express Igf2 mRNA. Using an Igf2
probe,
Figure 11: Results from trypsin-fiee cells using the Igf2 probe. (a) CelIs were manuaiiy
disocciated and placed on polarised slides. Probe seemed to accumulate on the nuclear membrane;
(b) Controls did not exhibit that same accumulation; (c) Cuitured cells were treated with the
cytoskeletal buffers and fixed. The Igf2 probe appeared to bind to parts of the membrane. possibly
the nuclear pores; (d) Controls did not drastically M e r from the experimentais. Magndkation
-2000~.
48 occasional nascent transcript signals were observed (Figure 12c); however, these resuits were rare
(refer to Table 4). Messenger RNA localized in the cytoplasm of the outgrowths, which was not
observed in the controls (Figure 12d).
The ectopiacentai cone outgrowths contained many cells which grew in three dimensions,
and oniy appeared in a monolayer at the periphery of the clumps. Having examined a large number
of these nuclei and compared them with the controls, it seemed as though the cytoplasm was
positively stained with IgPZ mRNA (Figure 13). Although cytoplasmic staining was not quantified
and levels of expression were not accurately assessed, it was very clear that no significant number
of nucfear sipals was detected in the flattened ceils of the experimental outgrowths.
3.4.3 Squashed Embryos
Day 8.5 embryos were squashed onto slides in order to force cells into a flattened state
resernbling a monolayer. Two treatments were tried: one included the 0.5% triton step for 2
minutes prior to fixation, and the other was immediate fixation with paraformaldehyde. No nuclear
signais were observed follow ing hybridization with the Igf2 probe. however the cytop lasrn
appeared to be positively stained (Figure 14). Background levels of autoflourescence greatly
obscured observations made of the nuclei, since much of the fluorochrome bound non-specifically
to the cytoplasmic components of ail ernbryonic cells.
Figure 12: Blastocysts were examined for RNA FISH. (a) Day 3.5 mouse emb~yos (arrow)
were cdtured. Blastocysts hatch in vitro and c e k extend and grow as shown by the DAPI DNA
stain. Mmcation -630x; @) A higher rnagnification of p r i m q giant cells, which constitue the
cells of the outgrowth, stained with DAPI; (c) Igf2 probe was utilized to look for transcripts. One
signal was observed in a giant ce11 (arrow); (d) Control nuclei (shown here overexposed) did not
have any detectable transcnpts. Magnification (b-d) - 1800~.
Figure 13: (a and b) Ectoplacenial cone outgrowths stained by DAPI; (c) Detection of nascent
uanscnpts using the IgfL probe resulted in no positive signals; (d) Control nuclei did not appear to
differ from the experimentals. Mapification - 1800~.
Figure 14: Squashed embryos were fixed after dissection with parafomaldehyde as shown in
(a); or treated with triton X-100 prior to fixation (b). (c) Using the Igf2 probe, no nuclear signals
were detected, very similar to the contml nuclei observed in (d). Magnification -2000~.
CfIAPTER 4
DISCUSSION
Fluorescent in situ hybridization is a very versatile technique most cornmonly used to
localize DNA sequences in metaphase and interphase cells, or to localize processed mRNA
expression patterns in cells. The application of this technique has increased dramatically in al1
fields of research for studies in cytogenetics, prenatal diagnosis, tumor biology, nuclear
organization, gene amplification and gene rnapping (Trask, 1991b). This thesis has attempted to
defme the optimal conditions for localizing the nascent pre-mRNA transcripts of imprinted genes in
the interphase nuclei of mouse embryonic cells. RNA FISH is relatively new in the world of
cytology, and theoretically provides one with the ability to examine active transcription of a wide
variety of genes in individual cells.
4.1 Success of RNA FISH on Cuitured Fibroblast CelIs
RNA FISH was successfidly achieved with the control gene, fibronectin, on a fibroblast
ce11 Iine. In order to examine the interphase nucleus, cells were grown to confluence where most
cells arrest in the G1 phase of the ce11 cycle, which was confirmed by DAPI staining of fibroblast
cells. Fibronectin is actively transcribed in the cell cycle, most in the G1 phase and least in the M
phase of the ce11 cycle (Stenman et al, 1977). It is also very abundantly expressed, its function
being to promote fibroblast chemotaxis, attachent, and spreading in culture, in tum making these
cells ideal to use for optimization of the conditions for RNA FISH. Fifty six per cent of all nuclei
(n=2952) were observed to contain two nuclear signals, which represent the biallelic expression
pattern of the fibronectin gene Studies by Perkinsin et al (1996) have shown that in cultured
fibroblast cells, fibronectin transcription is influenced by ce11 density, that is as confluence is
approached, the levels of fibronectin mRNA increase dramaticdly. The results presented here
support this published data since relatively large signais representing the nascent transcripts of
fibronectin were easily identified by RNA FISH. mRNA was also detected in the cytoplasm;
however, differences in positive signals here were observed depending on the length of treatment
with Triton X-100 prior to fixation. With shorter treatments, fibronectin mRNA was more readily
observed. Fibroblasts treated with Triton X-100 for up to ten minutes, which resulted in ceus with
a much reduced cytoplasmic component, had Iess intense staining and minimal rnRNA expression 56
57 could be observed in these nuclei.
Xing et al., (1995) demonstrated that the human fbactin DNA and RNA signals
overlapped in the fibroblast nucleus, however the extended tracks visualized by Lawrence et al.,
(1989) in the lymphoma ce11 line were not visualized and two small foci representing the DNA
signals k ing smailer than the RNA signals, could be observed. These signals appeared to be
"track-Iike" since they appeared to have some longitudinal axis when viewed through serial
sections. The mouse fibronectin signals observed in fibroblast nuclei also possessed some
longitudinal axis, usually 1.0 micron long. Specific tracks which extended to the nuclear
membrane were not observed,
4.2 Mouse Embryonic Cells
Cells retrieved for FISH analysis by dissection of day 7.5, and 8.5 embryos or by culture
of 10.5 day embryos proved to be unsuitable for the visuaiization of nascent transcripts of
impnnted genes. Problems resulting from the negative signals obtained may be the result of the
method of recovery of the nuclei, the cell types used, penetration of probe through the nuclear
membrane, and probe andlor target sequences.
4.2.1 Recovery of Nuciei
In order to anaiyze the transcription of a single gene, single cells must be obtained. The
original protocol for RNA FISH involved cytospinning non-adherent cells onto coated or polarised
slides. This method of low speed centrifugai force separates and deposits a monolayer of cells on
slides while rnaintaining their cellular integrity. The cells have a flatter morphology which is
essential for the detection of single-copy DNA or weak RNA signals. An optimal cenaifugal spced
would have to be determined if this apparatus were to be used, due to the destructive effects on cell
morphology which ultimately c m release cellular RNAases which exist in tissues. Despite this
problem, many investigators have successfUy used this apparatus (Johnson et al., 199 1 ; Panning
and Jaenisch., 1996; Panning et al., 1997). Elimination of this step may have contributed to the
lack of success with embryo cells.
Dissociation of tissues with trypsin presented difficulties in maintahhg the cell suspension
RNAase free. No signal was observed in crypsin treated embryo cells, while cells dissociated
mechanicdy displayed cytoplasmic hybridization. This suggested that the trypsin may have been
58 contaminated with RNAase. While fibmblasts were treated with trypsin to disperse them ont0
coverslips, they were ailowed to attach and grow for several &YS. Thus, the cells recovered from
any damage suffered during trypsin treatment, and cytoplasmiclnuclear equilibrium was restored
Culhiring of dissociated cells from dissections of day 10.5 embryos were not appropriate
for FIS& Although cells were given a pcriod of tirne to allow them to recover h m the dissection
and adhere to the gelaiinked coverslips, cells becarne mobilized throughout the washing phase of
the protocol, and for the cells which did remain attached, they were not flattened by their adherence
to the slide which made probe penetration very difficult The cytospinning apparatus would ideally
eliminate this problem with culaircd ceLls.
The method of dissociating cells from mouse embryos used by Wijerde et al., (1995) was
to disrupt fetal Iiver cells in PBS. Since only these cells were utiiïzed in their experiments, it may
be possible that liver cells dissociate easily in the saline solution, elirninating the need to treat
tissues with trypsin. The concentration of trypsin utilized in the f i t experiments attempted and
described in this thesis did include concentrations as low as 0.125 m g h l trypsin-EDTA, however
such a low concentration did not appear to be effwtive for the short incubation at 37 C. Panning et
al., (1997) have shown that trypsin treatment can be used to dissociate tissues of day 6.5 and 7.0
embryos with small volumes of trypsin (concentration not published) in multichambered slides.
However, individual day 7.5-8.5 ernbryos were recovered using the cytospin apparatus.
4.2.2 Ce11 types
Success using the RNA RSH protocol described in this thesis has been exclusively
performed by Lawrence and colleagues from 1989 to present, and Jaenisch and colleagues, 1996.
Other investigators (Huang and Spector, 199 1; Fakan and Nobis, 1978) have detected nascent
transcnpts in the interphase nucleus by different methods of in situ hybridization. However, an
underlying common theme in al1 of these published works is the fact that al1 cell types used are
cultured ce11 lines. Recently, Panning et al., (1997) have shown that Xist intron signals can be
detected using intron specific probes on embryos that were affixed to slides using a cytospin
apparatus.
Xist, the inactive specific transcript fkom the inactivated X chromosome, has been shown
to accumulate in the nucleus of female cells in human lymphoblast and fibroblast ce11 lines (Brown
et al., 1992) and mouse embryonic stem cells (Panning and Jaenisch., 1996) by RNA FISH. In
59 humans and mice, XLrt is abundandy expressed in female nuclei and its entire expression localizes
and coats the inactive X chromosome. Therefore the targets of the probe are many and the
transcripts are quite stable resuiting in large accumulations of positive signal visible by
fluorescence rnicroscopy. It has k e n recently shown that this accumulation of Xist RNA on the
inactive X chromosome is regulated by the stabilization of this transcript on the inactive X, raîher
tha . the stimulation of this gene's transcription from the inactive X in fibroblast ceils, ES ceils and
embryonic cells (Panning et al., 1997). Thus, the large FISH signals observed for Xiit do not
represent nascent transcripts only, but aiso processeci and targeted gene product.
The detection of primary transcripts in sin< are temporally and spatially associated with
recent transcription. Much information cm therefore be generated by studying the developmental
transcriptional regdation and chromatin dynarnics of an interphase nucleus as shown by Wijerde et
al., (1995). The five P-globin genes in humans are arranged in the order of their developmentai
expression. The transcriptional activation of these genes are dependent on their interaction with the
locus control region (LCR) located approximately 50 kb upstream. The transcriptional cornpetition
between these genes for the LCR is important to ensure that the proper P-globin gene is expressed
at the proper stage of development An alternate method of RNA FISH was used to detect the
primary transcripts of the gene in vivo using mouse fetal liver cells of a transgenic line carrying a
single copy of the complete human B-globin locus. Probes generated were oligonucleotides
complementary to intronic sequences of the p-globin gene, containing either biotin or digoxigenin
side chains. Antibody detection followed with as many as three or four amplification steps.
Results clearly illustrated that the LCR can only interact with one of the P-globin genes at a time in
nuclei by examining the nascent transcripts using this protocol (Wijerde et al., 1995).
4.2.3 Penetration of the NucIear Membrane
Accessibility into the nucleus quires the permeabilization of the nuclear membrane which
is usually achieved by treatments with proteinase K and pepsin (Dirks et al, 1993), streptolysin O
(Paillasson et al., 1997) or Triton X-10 prior to fixation (Lawrence et al, 1989; Panning and
Jaenisch, 1996) or after fixation (Huang and Spector, 1991). Treatment with 0.58 Triton X-100
prior to fixation with paraformaldehyde was the rnethod of permeablization for the positive
60 fibronectin signals observed here. However, puzzling results were observed when the triton
treatment was delayed untif after the furation step. Biallelic expression of fibronectin was not
observed if fixation preceded the Triton treatment. Although Huang and Spector (1991, 1996)
have reported that the nuclear signals could be detected by this mode of fixation, the target
sequences that were observed must be examined. Huang and Spector (1991) targeted the pre-
mRNA tmnscripts of the mouse c-fos gene after MH-3T3 cells grown in culture were s e m
starved, and then stimulated transcription of this gene by the addition of anisomycin, a protein
synthesis inhibitor. Each nucleus examined appeared to possess two active c-fos alleles. Recent
work by Huang and Spector (1996) used a transient and stable expression system of HeLa ceUs to
show that in these cells, transfected with various expression constructs by electroporation, the
nascent transcripts of these constructs are spatially assosociated with splicing factors. After
transfection, cells were grown for 36 hours to establish stable integration of the consmict. Cells
were then fixed with 4% parafonnaldehyde in PBS and treated with 0.5% Triton X-100. These
modes of ce11 pretreatment prior to hybridization suggest that the normal levels of gene
transcription are not as readily detectable without so.me transcriptional induction through
stimulation of transcription. Neither of these methods were attempted for the detection of
endogenous allelic expression for fibronectin or any imprinted genes.
4.2.4 Probes and Target Sequences
In order to target single genes and their nascent transcripts in the interphase nucleus, it is
essential to use probe fragment sizes which are from 150-300 bp in length and to use a high probe
concentration well above theoretical saturation Ievels recornrnended for the detection of mRNA in
the cytoplasm. The size and the intensity of the signal observed are proportional to the size of the
target sequence (Johnson et al, 1991), therefore larger targets are preferred. This is supported by
the success with the fibronectin cDNA probe, which is 20 kb long. Cornparison of the target
sequences of fibronectin and H l 9 might best exemplify this point. Biallelic expression of
fibronectin in mouse fibroblast cells is abundant and target sequences which are the unique nascent
transcripts of the gene can be as large as 48 kb. The fibronectin genes experiences aitemate
splicing to generate many functional cDNAs. The fibronectin probe utilized here therefore has
multiple targets to bind to. In contrast however, the H l 9 gene is only 3 kb long and if abundantly
transcribed in mouse embryonic cells, the actual target sequences are much less than those of
61 fibronectin. Although probe sizes are sirnilar for Hl9 and fibronectin. the function of such short
lengths is to facilitate the passage of the biotinylated probe into the nucleus. One laboratory
achieved nonstatistical detection of single copy DNA sequences as small as 2 kb and multiple
copies of RNA (Johnson et al, 1991), however hybridization to target sequences smailer than 10
kb c m be achieved, although the efficiency of hybridization obtained is often very low (20-4046)
with increased background making it more d i f f id t to see srnaller signals (Trask, 1991a).
Most positive results using RNA FISH employ probes which span very large sequences
such as 20 kb for the fibronectin gene in this work, 17 kb for the Xist gene (Brown et al., 1992),
20 kb for the human aibumin gene, and 40 kb for the human coilagen gene (Xing et al., 1995). Of
the imprinted genes utilized in this study, Igf2 would be appropriate due to its large size (19 kb);
however the H l9 gene, which encompasses 3 kb, would most Iikely be too srnall to ever detect
unless its endogenous expression is very high.
It was recently discovered that the Cot-l DNA added in the preparation of probes
throughout this thesis was not used appropriately. The function of Cot-1 DNA is to quench nay
repetitive sequences in the probe; however the Cot- 1 DNA and the probe were not denatured prior
to a one hour incubation ai 37 C to allow this quenching to occur. The probe was denatured prior
to the addition of the hybridization mix and then applied to slides. The Cot-1 DNA could have
bound to any repetitive sequences which exist in the nucleus, possibly binding to parts of the genes
which were being targeted. This might provide an altemate reason as to why no positive signals
were achieved when using the Igf2 probe on embryonic cells.
4.3 Blastocyst Odgrowths and Ectoplacental Cone Outgrowths
Culturing of blastocysts and ectoplacental cones takes advantage of the invasive nature of
the trophoblast cells. These cells tend to spread out and flatten on the tissue culture surface.
Moreover, they are polytene cells and would therefore contain multiple copies of genes at each
locus (Varmuza et ai.. 1988). If al1 of the copies are transcriptionally active, the RNA signal
should be amplified. However, each blastocyst could only yield a lirnited number of celis for the
length of tirne cultured. Ectoplacental cones grew vigorously as well but appeared to clump more
than the blastocyst outgrowths.
Although different methods of ceIl pretreatments were attempted, 1 found that these
treatments (See Results) did not aliow for the detection of any Igf2 pre-mRNA transcnpts in a large
62 number of giant cells examined. In the blastocysts outgrowth expriment (Figure 8), one signal
was obsexved; however it was only one in 256 nuclei examined. Due to the length of time that a
blastocyst is aven to attach and grow in vitro, cells which constitute the outgrowth may be
equivalent to cells of an early postimplantation embryo, which are part of the ectoplacental cone or
trophoblast, where expression of Igf2 is in fact monoallelic with strong patemal bias (Szabo and
Mann, 199%).
4.4 Methods of Improving the RNA FISH Technique
The technical problerns associated with RNA FISH appear to be numerous as described
above, and include the introduction of exogenous RNAases in attempts to dissociate tissues
adequately, and application of cells to slides in a thin monolayer, and permeabilization of cells to
allow for proper probe penetration. One possible means of eliminating some of the the technical
problerns in this technique would be the use of a cytospin apparatus. Panning et al., (1997) has
shown that cytospinning of cells is an essential step in the RNA FISH technique. Success has
been obtained with thÏs technique from other investigators using a four step fluorescence detection
amplification system enhancing the signal. Although reports show that this method also yields
p a t e r background levels, positive signals can be detected above background (Wijerde et al,
1995). Another means of achieving positive results is to use a single stranded probe generated by
PCR to target the nascent transcripts of the genes of interest (Barlow, personal communication). A
linear method of PCR is used to make single stranded probes which incorporate fluorescent
nucleotides. Using these PCR products as probes, positive signals can then be directly visualized
after an overnight hybridization since no ampmcation step is involved. The use of radioactive
probes instead of fluorochromes in high resoiution autoradiography using electron microscopy has
been shown to successfully work for a number of genes, particularly Snrpns, hnRNAs, and
nuclear antigens and poly (A)+ RNAs (Fakan and Nobis. 1978; Lehner et al., 1986).
4.5 Monoallelic Expression of Imprinted Genes
Monoallelic expression of imprinted genes is responsible for the inability of uniparental
mafnmalian embryos to develop normally and for the abnormal phenotypic effects observed in
embryos with chromosome disornies. Some of the imprinted genes identified to date are growth
factors, tumor or growth suppressors, cell cycle regulators, or non protein coding RNAs.
63 Although all irnpnnted genes are characterized by allele-specific expression, the regulaîion of the
imprinted expression is very compiex. Examining the allelic expression patterns of irnprinted
genes in a wide array of tissues can d o w for the identification of the impriut status and provide a
pattern of imprinted gene expression during development Detection of primary transcripts in situ
can provide a very accurate time frame to determine when n o d gene expression switches from a
biallelic mode to monoailelic expression.
The mechanism in which one actively transcribing allele is silenced has not been
detennined. Studies on androgenetic, gynogenetic and parthenogenetic preimplantation mouse
embryos have show that the inactivation of imprinted genes occur postfertilization, most likely
postimplantation, and that the time at which one allele is silenced is not necessarily the same the at
which the zygotic genome is activated (Latham et al., 1994). These authors propose that
chromosomes which contain imprinted genes contain an imprint rnark which is recognized at a tirne
point in development by regulatory factors which are expressed in cells. The imprint rnark may be
set down during gametogenesis or during the early posdertilization period. These regulatory
factors can then recognize the mark and selectively inactivate one parental allele resulting in the
appropriate monoallelic expression of that specifc imprinted gene.
The establishment, recognition and maintenance of imprinting are the focus of numerous
laboratories which are attempting to identiQ what the imprint mark is and how it is maintained.
Studies have shown gene expression is biallelic for most genes in the gedine, and monoallelic
expression occurs later in development (Szabo and Mann, 1995b; Latham et al, 1994; Mann et al,
1995). DNA methylation has been shown to be important in the maintence of monoallelic
expression (Li et al., 1993), and differences between the replication timing of homologous
chromosomes suggests that differences in DNA replication or chromatin structure may be common
to genes which are monoallelically expressed (Kitsberg et al., 1993).
The optimization of the RNA FISH technique could have many practical uses in research
and prenatal diagnosis. The identification of new, unknown imprinted genes could be simplified
by using RNA FISH on various tissues of the developing mouse embryo and looking for a switch
from biallelic to monoallelic expression. As well, this technique could be used in clinical diagnosis
of human genetic disorders such as the Beckwith-Weidemann syndrome which has inappropnate
expression of the Igf2 and Hl9 genes from the normaliy silent alleles.
64 4.6 The interphase nucleus and imprinted genes
Attempts to undentand the inactivation of the X chromosome in female mammals has
successfully bridged two fields of nsearch: understanding the dynamics of the interphase nucleus
and the complex gene regulation of the inactivated X. Using RNA FISH, various authors
(Clemson et al., 1996; Lee et al, 1996; Panning and Jaenisch, 1996; Panning et al., 1997) have
been able to visually identiQ which chromosomes transcribe the Xisr transcripts, where these
transcnpts localize, and how this transcript may act as an imprinting control centre to silence other
genes on the inactive X chromosome. In addition, DNA FISH was successfully used to discover
clifferences in replication timing of imprinted gene regions (Kitsberg et al, 1993). The interphase
nucleus provides researchers with tremendous amounts of information to understand how complex
gene regulation occurs. Much work on the interphase nucleus has focused on the organization of
the nucleus, how nascent transcripts are spliced and whether the transcripts are associated with
splicing factors which are concentrated in 20-40 distinct dornains called speckles (Misteli et al.,
1997). Optimizing methods of preparing individual nuclei (other than ce11 lines), and preparation
of suitable probes, as recently shown by Panning et al., (1997), may facilitate studies to identify
when one allele of an imprinted gene is transcriptionally silenced and whether monoallelic
expression persists through development.
SUMMARY
1. The technique of RNA FISH requires that cells are Battened either by growth in culture, as
achieved by cell lines or by cytospinning.
2. Tissues must be disaggregated without any destructive effects on the cellts morphology and
without the introduction of any exogenous RNAases or release of any endogenous RNAases.
3. Probe size and nicked fragment lengths are of vital importance in gaining access into the
nucleus. Nicked fragments should range from 150-500 bp and the target gene sequences should
ideally be greater that 10 kb.
4. The pre-mRNA which is king targeted must be very abundantly transcribed in the nucieus at
the t h e of fixation.
CONCLUSION
RNA FISH allows for the direct visualization into the interphase nucleus, in that many
studies undertaken thus far have allowed for a better understanding of what is occurrïng in the
uncompartmentaiized nucleus. The nucleus is a dynamic, yet highly structured entity, which is
under strict regulation to ensure that the DNA which it contains is properly replicated and
transcribed, and the RNA products are exported to the cytoplasm. RNA FISH should be capable
of identifylng the time at which irnprinted gens are silenced and if in fact they are globally silenced
in a tissue, or to veriQ if these imprinted genes exhibit le* expression. RNA FISH could
identifi whether any cells remain biallelic in a tissue which would otherwise be undempresented
by other methods which detect RNA expression, such as RT-PCR. This technique can be
extended to many other tissues and systerns to examine when genes are actively king transcribeb
Currently, most results are based on the mRNA expression patterns which are not precisely
indicative of the transcription of a gene since some mRNA can have a relatively long half life and
exist in the cytoplasm long after the transcription of the gene has ceased. The technical problems
associated with RNA FISH presently impede the widespread use of this precise technique to study
the transcriptional activation of any gene. By overcoming such obstacles, much can be learned
about imprinted genes in the early embryonic development of the mouse.
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