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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