reverse genetics
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Reverse Genetics
J. Lieb
April 19, 2006
Wild-type Bmp7 -/-
Mouse Embryos
Remember Forward Genetics? :
Phenotype Gene
or
Mutations First Molecular Analysis Second
Reverse Genetics
Gene Phenotypeor
Molecular Analysis First Mutations Second
Example Uses:
• Understand the function of a gene homolog characterized in another organism
• Understand the function of individual amino acids or protein domains.
• Create “conditional alleles” of a gene.
Review of Last Lecture
"Model" Organisms in Biology
What allows us to use them?1. All organisms share similar cellular machinery2. All animals use this machinery in similar ways to direct embryonic
development
Why use them?Perform controlled experiments on a large number of samples to
learn about:• Basic Molecular Mechanisms (Yeast Cell Cycle: Cancer)• Mechanisms of Genetics (Mendel’s Peas; Fruit Flies)• Embryonic Development (C. elegans, Sea Urchins; Mice)
What does one look for when choosing a model?
Fast, cheap, easy to observe and manipulate, and has the feature you want to study
TransgenicsUsing the power of molecular biology to isolate and clone the DNA of our choice, and then to express it in a controlled manner in the organism of choice.
Review of Last Lecture
Why?
• To study the role of a gene in development
• To see where a gene is expressed
• To understand what happens when a gene is misregulated
• To “cover” a genetic defect
GENE TARGETTING
• Dirigido a un gen específico.
• Silenciamiento del gen a nivel del RNA.
• Reemplazo o modificación del gen.
SILENCIAMIENTO DEL GEN
• Acción a nivel de mRNA (evitando que sea traducido)
Mecanismos:
• Oligonucleótidos Antisentido : oligonucleótido con la secuencia complementaria al mRNA blanco (target )
• Ribozimas: RNAs con actividad catalítica, se une y corta al mRNA blanco (target)
• iRNA : RNA de interferencia
Oligonucleótidos antisentido (Antisense oligos)
• DNA o RNA de hebra simple
• Secuencia complementaria al mRNA target
• Mecanismo de acción : formación de doble cadena y:
Bloqueo del Inicio / elongación de traducción
Alteración del procesamiento del mRNA : si está dirigido a la secuencia limite exón/intrón bloquea la función de los snRNAs que intervienen durante el splicing
Alteración de la estabilidad / vida media del mRNA: si el oligo está dirigido a una secuencia en el 3´ UTR y que impida la formación de hairpins propios del mRNA que lo estabilizen; por ejemplo: mRNAs de histonas no tienen poly A, pero sí forman hairpins en el 3´ UTR.
En vertebrados existen dos tipos de histonas:
- Histonas independientes de replicación : sus mRNAs son polyadenilados
- Histonas dependientes de replicación, sus mRNAs: - presentan cola de polyA - presentan una estructura de tipo stem-loop en el 3´UTR - presenta una secuencia de reconocimiento para el procesamiento o maduración del extremo 3´.
El oligo Antisense puede estar dirigido a:
- bloquear la formación del stem-loop
- bloquear la interacción del mRNA con el snRNA.
mRNA de histonas dependientes de replicación
Antisense dirigido a bloquear la formación de los hairpins
El pre-mRNA de HIV integrado debe formar 2 hairpins (TAR) necesarios para la unión de la proteína TAT al RNA
Oligonucleótidos con mayor tiempo de vida media
PNAs : Peptide-nucleic acids
• Esqueleto formado por enlaces peptídicos : estabilidad, mayor tiempo de vida media
• Bases nitrogenadas : confieren especificidad de acción
Principal desventaja :
- toxicidad
RIBOZIMAS
• RNAs con actividad catalítica, se une y corta al mRNA blanco (target)
• Estructura estable : hammerhead
• Pueden ser quimicamente modificados los extremos para incrementar su vida media
5’- - c g g a g u c a c u u c g - - 3’ mRNA 3’ G C C U C A U G A A G C 5’
AIAGCGGCG
GCCGC
UC
C
G U
AGU
AG
U
Ribozima
Mecanismo de corte por ribozima
TRANSGENIA
Conceptos
Diferencia entre clonación y transgenia
•Identification of gene function
•Generation of animal models of diseases
•Drug validation
•Cell and organ research
Others…
WHY?
How many genes are there in mammalian cells?
E. coli 4.6 Mb 4,288 genesS. cerevisiae 13.5 Mb 6,034 genesD. melanogaster 165 Mb 12,000 genesC. elegans 97 Mb 19,099 genesH. Sapiens 3,300 Mb 40,000 genes
Genome project was completed in 2002 (still regions that are unclear)
Gene expression profiling (Exon profiling)
Phenotypes
Studied on the mechanismof gene expression.
Genomics and proteomics
Transgenic technologies
MAMMALIAN EMBRYO MANIPULATIONS
Animal models?
Mouse and rats Research
Commercial uses:Farm animals
Improve health, Cure diseases , others?
In theory, all mammalian embryos can be used.
Human
The MOUSE Life span: approx. 2.5 years
Gestation : 21 days
Litter size: 8 to 12
Generation time: three months
Several inbred and outbred strains
Genomic database
Most advance genetic technologies
Cost per mouse/$2 to 24$
Housing cost
Over 90% identical to human genome
Large enough for physiological studies
MAMMALIAN EMBRYO MANIPULATIONS
NON-TRANSGENIC
TRANSGENIC
No modifications to the genome.
Modifications to the genome.
Germline mutations Somatic cell mutations
Natural or in vitro
fertilization
Spermatozoide
Oocyte
Enucleation
Nuclear transfer and cloning
Transgenic Animals by pronucleus injection
-Genotyping of genetic diseases -Freezing and storing -Embryo cleavage and cloning
Transgenic and embryo manipulation in mammals
Morula
Blastocyst
- Isolaton of embryonic stem cells - Gene targeting
Primary cells culture
Cell therapy Neuronal cells
Muscle cells
Epithelial cells
Stem cells
1
23
4
in vitro Différentiation
5
6
7
8
Spermatozoide
Oocyte
6hrs 18hrs 36hrs 48hrs
Fertilizedegg
ZygoteMorula
Blastocyst
3 daysImplantation
4 days
Spermatozoide
Oocyte Fertilizedegg Morula Blastocyst
-NuclearTransfer-Cloning
Transgenic By Microinjection
Freezing Splitting InfectionGenotyping
-Embryonic stemCells
-Gene targeting
Oocyte
For cloning
Nuclear Transfer Technology
Nucleus of stem cells or others
Nuclear Transfer Technology
Usage?
Cloning of: - Valuable cheptel (farm animals) - Endangered species- Basic research in stem cell tech.- Others?
Distal>100kb
ProximalApprox. 1kb
Promoter TranscriptionUnit
Coding and uncoding sequencesfrom 1kb to >200kb
Gene Locus: Includes both the promoter and the transcription unit!
Transgenic Technology
Promoter
Temporal and spatialexpression
cDNA Intron
Gene of interest
AAAA
AAAA
Transgene
<1kb to >200kb
Transgenic Technology
Promoter
cDNAGain of Function
1- Tissue specific (brain, liver, muscle …)2- Ubiquitous
3- Inducible (tetracyclin, interferon...
Loss of Function
- wild-type gene- mutant
- dominant negative- antisense- ribozyme
Identification of the important features of a promoter.
A: Comparison of sequences: Increasing uses
B: Use of reporter genes:
Databases: Genebank and others.
Gene xPromoter of gene x
Promoter of gene x Reporter gene - B galactosidase- Luciferase- GFP- others….
Transgenic Technology
6hrs
MosaicDNA
Transgenic Technology
6hrs
FVB/N
XX
Ste.
CD1
Fos
Fos
Reimplantationin the oviduct
Pregnancy
Fos
Transgenic Technology
Advantages Disadvantages
- Short time to produce(21 days)
- High level of expression- Cheaper cost- Simple vectors
- Random integration- Multiple integration sites- Each animal has
different genotype
Embryonic stem Cellsand
Gene targeting
Gene knock-out and Gene Knock-in
Knock-out Mice
Homologous recombination
Embryonic Stem cells
Oliver SmithtiesMario Capecchi
Neomycin
Neomy
mycinX
Approx. 500 bp
Neomy
mycinX
In chromatin Episomal
Homologous recombination
1- Length of homologous sequences
Hom. Rec. Efficiency
Base pair
25bp 2000bp
2- Isogenic DNA
NEOMYCIN
X X
Gene targeting
NEOMYCIN
Total 4 Kbp (each arm not less than 500bp)Delete coding sequencesChange reading frame
Transcription of Neo in antisense direction
Embryonic Stem cells Blastocyst 3 days
Inner Cell Mass
1-totipotent
2-tissue culture
3-Transfectable
4-Selection
5- DifferentiationIn vitro
129/sv(agouti)
C57Bl/6Black
Foster mother2 day-pregnant
X
X
1 WT 2 Hetero 1 Homo
Site specific recombination: Cre-Lox system, Flp recombination
From P1 phage
Cre recombinase Lox site(approx 30 bp)
Excise and integrate DNA
Excisea b c
a bc
a c
b
Integratege
f
e f g
Cre lox in the mouse
- Temporal and spatial targeting- knock-in- single point mutation- translocation
Brain
KO in the brain only
KO in the adult only
Adult-P
X
Lox mouse
Cre mouse
Day 50 (end of Week 7)
What about transgenics in mammals?
A relatively new (1980s)- molecular approachRecipe for a gene "knockout“ in mice:
Step 1Problem: Find a cell line that can grow in tissue culture but also retains the potential to become part of a real embryo.
Solution- Embryonic stem cells
ES or Embryonic stem cells:
Blastocyst-stage cellsthat have been coaxedand coddled intogrowing in culture
Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, leading to production of chimeras
A mouse with“3 parents”
Adding a gene: Production of Transgenic Mice
Production of Transgenic Mice 2
Embryonic stem cells (ES cells) are then incorporated into blastocysts, with the hope that they “go germline”.
If so, a line is created
Production of Transgenic Mice 3
Production of Transgenic Mice 3
OK, we've added a gene (Transgenics).
Now, we want to make a KO(Reverse Genetics)
Mario Capecchi
mRNA
Gene X
mRNA
Gene X
A normal cell has two copies of gene X:
No mRNA
Gene X
mRNA
Gene X
Neo resistance gene
Scientists use homologous recombination to insert gene for resistance to the drug neomycin into the middle of one of the copies of gene X, destroying its function.
Recipe to "knockout" a gene: Step 2
Oliver Smithies(UNC)
Recombinant ES cells can then be selected in culture.
Technique for Gene Targeting (1 of 3)
Technique for Gene Targeting (2 of 3)
Technique for Gene Targeting (3 of 3)
Morphological Analysis of Bmp7 Knockout Mice
Morphological Analysis of Bmp7 Knockout Mice
www.hgu.mrc.ac.uk/Research/Devgen/Cysfib/julia.htm www.cf.ac.uk/biosi/staff/jacob/teaching/ionchan/cftr.jpg
Mouse models of human diseasehelp us to design and test new treatments
A transgenic humanTreated for SCID
Other Reverse Genetic Approaches
• Site-directed mutagenesis
• RNAi
• Chemicals (Chemical Genetics)
Site-directed mutagenesis
Gene Replacement
RNA Interference
Method 1 Method 2 Method 3
Mechanism of RNAi
iRNA RNA de interferencia
RISC: RNA induced silencing complex
RNAi en la expresión de GFP
Fig 4. Small interfering RNAs vs Small temporal RNAs
Forward and Reverse "Chemical Genetics"
REEMPLAZO DE GENES
• Integración del fragmento de DNA en el genoma del hospedero- en el sitio del gen homólogo o en sitios al azar. La integración en sitios al azar es más frecuente.
• Si existen secuencias homólogas en el genoma puede haber recombinación homóloga. Se da el reemplazo del gen específico del hospedero (Knock-out).
• En la integración al azar, la expresión del gen puede verse afectada según el lugar de inserción, puede interrumpir o afectar a otros genes.
• Células somáticas vs. Células germinales - transgénico.
GENERACION DE ANIMALES TRANSGENICOS
1. Cultivo in vitro de células troncales de embrión (Embryonic stem- ES).
2. Preparación del gen a insertar. (Ej: BMP7 clonado, es interrumpido por el gen de resistencia a Neomicina como marcador de selección).
3. Transferencia del DNA exógeno a las células.
4. Selección de las células donde ha ocurrido el reemplazo del gen utilizando el marcador de selección (resistencia a Neomicina)
5. Las células seleccionadas se insertan en un embrión nuevo, el cual es colocado al útero.
6. Progenie resultante: quimeras con algunos tejidos heterocigotes y otros tejidos silvestres.
7. El cruce de una quimera con un animal silvestre dará una progenie heterocigote
(BMP7+/BMP7-) si las cells ES modificadas han contribuido a la linea germinal.
8. Del cruce entre los heterocigotes, aprox. 25% de la progenie será homocigote
trangénico BMP7- /BMP7-
Ejemplo: knock-out del gen BMP-7
MARCADORES DE SELECCIÓN
• Selección positiva : célula + marcador insertado la célula vive
• Selección negativa: célula + marcador insertado la célula muere
Por ej:
• Gen de resistencia a Neomicina (marcador de selección positiva)
Cell + Neor sobrevive
• Gen Timidina Kinasa del HSV (marcador de selección negativa)
Cell + TK HSV muere
en presencia de Neomicina
en presencia de ganciclovir
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