Transcript Heather Cross - Virginia Commonwealth University

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
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.
LoxP
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
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?
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