Propagation of the Treacher Collins Syndrome Mouse Model

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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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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 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

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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/LoxP System

• 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
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Heterozygous for Cre and loxP
1A5-N-1 or 1A5-N-3
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Our Timed Mating Scheme
These are the homozygous knockout mice that 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 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.

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Tcof1 -/- homozygotes Vs. Wildtype
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Tcof1 -/- homozygotes Vs. Wildtype
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Mouse Embryos with Tek Staining
Tcof1 -/- homozygotes
Wildtype Embryos
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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 to Treacher 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 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

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The reason for the fragments in gels
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.
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Reading the Gel
Cre line
Heterozygous for loxP
Homozygous for loxP
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 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

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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.

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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!

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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/?p287
• http//ghr.nlm.nih.gov/genetcof1
• http//www.nlm.nih.gov/medlineplus/article/001659.
  htm
• http//www.treachercollins.org/main.html