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Propagation of the Treacher Collins Syndrome Mouse Model By Heather Dawn Cross Mentor: Dr. Rita Shiang Grad Student: Michelle Holser.

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Presentation on theme: "Propagation of the Treacher Collins Syndrome Mouse Model By Heather Dawn Cross Mentor: Dr. Rita Shiang Grad Student: Michelle Holser."— Presentation transcript:

1 Propagation of the Treacher Collins Syndrome Mouse Model By Heather Dawn Cross Mentor: Dr. Rita Shiang Grad Student: Michelle Holser

2 Working in the lab; Loading a gel

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

4 Michelle; my grad student mentor

5 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

6 The Genetics The Treacher Collins gene, identified as TCOF1, is located on the 5 th chromosome, and codes for the protein treacle. The disorder is autosomal dominant. The protein, treacle, is involved in craniofacial development in embryos

7 Treacher Collins Gene

8 Cre/LoxP System Cre- cyclization recombination, loxP - locus of X-over P1,34 base pairs where Cre can bind to recombine the DNA

9 Basic Idea

10 Our Timed Mating Scheme ♀ ♂ Heterozygous for Cre and loxP 1A5-N-1 or 1A5-N-3

11 Our Timed Mating Scheme ♀ ♂ These are the homozygous knockout mice that we are studying.

12 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

13 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.

14 Tcof1 -/- homozygotes Vs. Wildtype

15

16 Mouse Embryos with Tek Staining Tcof1 -/- homozygotesWildtype Embryos +/+ 8.5? -/- 8.5? +/ /- 10.5

17 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.

18 Transfer Mating ♂ ♀ ♂♂ ♀♀ 1A5-N-1 or 1A5-N-3 generation 1 B6

19 ♂♂ ♀♀ 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 10 th generation of mice.

20 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

21 The reason for the fragments in gels Exon 1 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

22 Reading the Gel Cre line Heterozygous for loxP Homozygous for loxP Homozygous for wild type

23 Embryo Gel

24 An Actual Gel, Pups

25 Actual Gel, Embryos

26 Other Gels

27 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

28 Pictures of Me at Work

29 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

30 Something New I was honored enough to see something that Dr. Shiang has never seen before in a timed dissection. On July 19 th 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.

31 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!

32 ANY QUESTIONS? Thank you for your time and attention!

33 Sites used for presentation 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://phenome.jax.org/pub- cgi/phenome/mpdcgi?rtn=docs/home htmhttp://www.nlm.nih.gov/medlineplus/article/ htmhttp://www.nlm.nih.gov/medlineplus/article/ htmhttp://www.nlm.nih.gov/medlineplus/article/ htm


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