Propagation of the Treacher Collins Syndrome Mouse Model By Heather Dawn Cross Mentor: Dr. Rita...

Propagation of the Treacher Collins Syndrome Mouse

Model

By Heather Dawn CrossBy Heather Dawn Cross

Mentor: Dr. Rita ShiangMentor: Dr. Rita Shiang

Grad Student: Michelle HolserGrad Student: Michelle Holser

Working in the lab; Loading a gel

Dr. Rita Shiang, my mentor, looking over some pictures I took.

Michelle; my grad student mentor

What is Treacher Collins

• A rare genetic disorder

• Characterized by– Abnormal or absent

external ear– Hearing loss– Very small lower jaw– Defect in lower eye– Cleft palate– Breathing problems– Down slanting eyes

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

Treacher Collins Gene

Cre/LoxP System

• Cre- cyclization recombination,

• loxP - locus of X-over P1,34 base pairs where Cre can bind to recombine the DNA

Basic Idea

Our Timed Mating Scheme ♀

♂

Heterozygous for Cre and loxP 1A5-N-1 or

1A5-N-3

Our Timed Mating Scheme♀

♂

These are the homozygous

knockout mice that we are studying.

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

Embryos• The yolk sac from each embryo is

genotyped to separate out the homozygous knockouts, heterozygous knockouts and wild types.

• The embryos are collected until there are some homozygous knockouts for each developmental stage.

• At this point, various experiments can be performed with the embryos.

Tcof1 -/- homozygotes Vs. Wildtype

Tcof1 -/- homozygotes Vs. Wildtype

Mouse Embryos with Tek Staining

Tcof1 -/- homozygotes Wildtype Embryos

+/+ 8.5?-/- 8.5?

+/+ 10.5-/- 10.5

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 to Treacher Collins Syndrome.

• It takes 10 generations for each mouse line to be considered transferred.

Transfer Mating♂ ♀

♂♂ ♀

♀

1A5-N-1 or 1A5-N-3

generation 1 B6

♂♂ ♀

♀

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.

Genotyping• Each pup or embryo goes through

genotyping

• 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

The reason for the fragments in gels

Exon 1

Exon 1LoxP LoxP

The amplified Cre fragment being the longest would run the slowest in a gel

The addition of the LoxP sites makes the PCR fragment of the gene longer; so when separated in a gel it would be slower than the natural gene.

Cre

Reading the Gel

Cre line

Heterozygous Heterozygous for for loxPloxP

Homozygous Homozygous for for loxPloxP

Homozygous Homozygous for wild typefor wild type

Embryo Gel

An Actual Gel, Pups

Actual Gel, Embryos

Other Gels

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

Pictures of Me at Work

What Happened

• During the summer we were able to collect mice from– 8.5 dpc – 9.5 dpc– 10.5 dpc– 11.5 dpc

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

• This resulted in the inability to continue on with any further experimentation

Something New• I was honored enough to see something

that Dr. Shiang has never seen before in a timed dissection.

• On July 19th while doing a 9.5 dpc dissection, I found a set of identical twins in the embryos.

• Normally each embryo has its own “bead” and yolk sac but there were 2 embryos in one “bead” sharing a yolk sac.

This summer• This summer I learned a lot about

research– 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 mice do not always get pregnant when you want them to!

ANY QUESTIONS?ANY QUESTIONS?

Thank you for your time and Thank you for your time and attention!attention!

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