heather cross treacher collins syndrome-hdc

7/30/2019 Heather Cross Treacher Collins Syndrome-HDC

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Propagation of the TreacherCollins Syndrome Mouse Model

By Heather Dawn Cross

Mentor: Dr. Rita Shiang

Grad Student: Michelle Holser


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Working in the lab; Loading a gel


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Dr. Rita Shiang, my mentor, looking

over some pictures I took.


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Michelle; my grad student mentor


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What is Treacher Collins

A rare geneticdisorder

Characterized by

Abnormal or absentexternal ear

Hearing loss

Very small lower jaw

Defect in lower eye

Cleft palate

Breathing problems

Down slanting eyes
http://www.treachercollins.org/pictures.html


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The Genetics

The Treacher Collins gene, identified as

TCOF1, is located on the 5th chromosome,

and codes for the protein treacle.

The disorder is autosomal dominant.

The protein, treacle, is involved in

craniofacial development in embryos


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Treacher Collins Gene


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Cre/LoxPSystem

Cre- cyclization recombination,

loxP- locus of X-over P1,34 base pairs

where Cre can bind to recombine the DNA


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Basic Idea


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Our Timed Mating Scheme

Heterozygous

forCre and loxP 1A5-N-1 or1A5-N-3


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Our Timed Mating Scheme

These are the

homozygous

knockout micethat we are studying.


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The Timed Matings

These timed matings are used for

dissections to characterize the model.

They are dissected at specific time points

8.5 dpc

9.5 dpc

10.5 dpc

11.5 dpc

12.5 dpc


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Embryos

The yolk sac from each embryo isgenotyped to separate out thehomozygous knockouts, heterozygousknockouts and wild types.

The embryos are collected until there aresome homozygous knockouts for eachdevelopmental stage.

At this point, various experiments can beperformed with the embryos.


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Tcof1 -/- homozygotes Vs. Wildtype


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Tcof1 -/- homozygotes Vs. Wildtype


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Mouse Embryos with Tek StainingTcof1 -/- homozygotes Wildtype Embryos

+/+ 8.5?-/- 8.5?

+/+ 10.5-/- 10.5


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B6 Background Transfer

Transferring the 1A5-N-1 and the 1A5-N-3

lines from a SV/J to a B6 mouse genetic

background, because the B6 mice show a

phenotype similar but more severe toTreacher Collins Syndrome.

It takes 10 generations for each mouse

line to be considered transferred.


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Transfer Mating

1A5-N-1 or

1A5-N-3

generation 1 B6


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N-1-B6-02 or N-3-B6-02

generation 2

We keep only the positive males to be mated to female B6 to

continue the background transfer. This will continue till we reach

the 10th generation of mice.


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Genotyping

Each pup or embryo goes throughgenotyping

The DNA is extracted from an ear punch

or the yolk sac tissue

The DNA is amplified by PCR

The DNA is then run on a gel forming a set

of lines that defines the genotype


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The reason for the fragments in

gels

Exon 1

Exon 1LoxP LoxP

The amplifiedCre

fragment being thelongest would run the slowest in a gel

The addition of the LoxPsites makes the

PCR fragment of the gene longer; so when

separated in a gel it would be slower than

the natural gene.

Cre


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Reading the Gel

Creline

Heterozygous

forloxP

Homozygous

forloxP

Homozygous

for wild type


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Embryo Gel


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An Actual Gel, Pups


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Actual Gel, Embryos


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Other Gels


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My Role

My responsibilities include helping with:

the general care of the mice

keeping the mouse lines alive and properly

mated genotyping the pups and embryos

DNA extractions

PCR

gels

timed mating dissections Processing embryos


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Pictures of Me at Work


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What Happened

During the summer we were able to collect micefrom 8.5 dpc

9.5 dpc

10.5 dpc

11.5 dpc

Due to 2 different false pregnancies in the micewe were unable to collect mice from the 12.5dpc

This resulted in the inability tocontinue on with any further

experimentation


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Something New I was honored enough to see something

that Dr. Shiang has never seen before in atimed dissection.

On July 19th while doing a 9.5 dpc

dissection, I found a set of identical twinsin the embryos.

Normally each embryo has its own bead

and yolk sac but there were 2 embryos inone bead sharing a yolk sac.


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This summer

This summer I learned a lot aboutresearch

It doesn't always work

Some days are exciting and some are not

Working in a lab group is a unique experience Research is something that I could see myself

doing in the future

The m ice do not always get

pregnant when you want them to!


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ANY QUESTIONS?

Thank you for your time and

attention!


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Sites used for presentation

http://mouseworksonline.com/

http://phenome.jax.org/pub-

cgi/phenome/mpdcgi?rtn=docs/home

http://www.scq.ubc.ca/?p=287

http://ghr.nlm.nih.gov/gene=tcof1

http://www.nlm.nih.gov/medlineplus/article/001659.htm

http://www.treachercollins.org/main.html
http://mouseworksonline.com/http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/homehttp://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/homehttp://www.scq.ubc.ca/?p=287http://ghr.nlm.nih.gov/gene=tcof1http://www.nlm.nih.gov/medlineplus/article/001659.htmhttp://www.nlm.nih.gov/medlineplus/article/001659.htmhttp://www.treachercollins.org/main.htmlhttp://www.treachercollins.org/main.htmlhttp://www.nlm.nih.gov/medlineplus/article/001659.htmhttp://www.nlm.nih.gov/medlineplus/article/001659.htmhttp://ghr.nlm.nih.gov/gene=tcof1http://www.scq.ubc.ca/?p=287http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/homehttp://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/homehttp://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/homehttp://mouseworksonline.com/

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