Propagation of the Treacher Collins Syndrome Mouse Model

By Heather Dawn Cross Mentor: Dr. Rita Shiang Grad 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.

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

Transfer Mating

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

Cre LoxP Exon 1 LoxP

Exon 1

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.

Reading the Gel

Cre line Heterozygous for loxP Homozygous for loxP Homozygous for 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

 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.

Something New

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?
Thank you for your time and attention!

Sites used for presentation

http://mouseworksonline.com/ http://phenome.jax.org/pubcgi/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