Page 63 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
63
repositories for the scientific community to build on the information provided by the primary phenotypic screen for a more
detailed and specific analysis. To date over 700 lines have been produced by the Sanger MGP. To ensure the quality of this
resource, new colonies are subject to a panel of quality control (QC) PCR-based tests to confirm the targeting of the endogenous
allele, structure of the cassette, absence of additional insertions and for the presence of the 3 LoxP site. Approximately 86% of
lines passed the targeting and cassette structure tests; while 96% had the expected 3 LoxP site. The characteristics of the QC-
failed lines are discussed and should act as a guide for researchers generating their own lines on what potential issues may exist
and how to detect them. Many of the issues observed in the mouse lines may be derived from mixed clone populations which
were not detected using the mainly PCR-based primary screen on the ES cells. Therefore, it is vital to check all heterozygous
chimera progeny for each colony with all tests to detect these events and prevent them from propagating through the colony.
84
An albino C57BL/6N strain for EUCOMM/KOMP mouse generation in a pure genetic background. Ed Ryder
,
Diane
Gleeson, Thomas Keane, Debarati Sethi, Sapna Vyas, Hannah Wardle-Jones, James Bussell, Richard Houghton, Jennifer
Salisbury, David Adams, Ramiro Ramirez-Solis, Sanger Mouse Genetics Project. Wellcome Trust Sanger Institute, Cambridge,
United Kingdom.
The Wellcome Trust Sanger Institute Mouse Genetics Project (MGP) generates 160 strains from the international KOMP and
EUCOMM targeted mutant ES cell resource per year. The phenotypic consequences of the modified alleles are characterized in a
comprehensive primary screen. All mice are now produced on a pure C57BL6/N background, in line with the emerging
International Mouse Phenotyping Consortium (IMPC) and associated programmes including the NIH-funded KOMP2 project.
Normally, to preserve the purity of the genetic background, all chimeras are mated to black C57BL/6N mice. Therefore colour
selection cannot be used to detect germ-line transmission (GLT), and consequently all G1 mice have to be genotyped. Here, we
introduce a C57BL/6N albino strain which restores the ability to score GLT by coat colour of the chimera progeny, greatly
decreasing costs and effort, and improving animal welfare. The new strain originated from a mutation of the Tyrosinase locus in
an ES clone used to generate one of the mutant strains in the MGP. This mutation consists of a 14.3Kb deletion spanning 940bp
5
of the start of exon 1 to 3.75kb 3 of exon 2. Genome sequencing of the two albino founder mice of the colony is now underway
to determine whether any large deleterious mutations are present which could limit its use in future downstream applications.
85
The transcription factors
Ets1
and
Sox10
interact during murine melanocyte development. Amy Saldana-Caboverde
,
Lidia Kos. Biological Sciences, Florida International University, Miami, FL.
Melanocytes, the pigment-producing cells, are derived from a population of pluripotent migratory cells known as the neural
crest (NC) that delaminate from the dorsal aspect of the neural tube during vertebrate development. Many of the genes required
for the specification of melanocytes from the NC have been identified through the study of mouse pigmentation mutants. Mice
carrying a deletion of the transcription factor
Ets1
were recently shown to exhibit hypopigmentation; nevertheless, the function
of
Ets1
in melanocyte development is unknown. In the mouse embryo,
Ets1
is widely expressed in developing organs and tissues,
including the NC, and is required for the endogenous expression of the melanocyte-specific transcription factor
Sox10
in the
chick cranial NC. We aim to establish the temporal requirement and role of
Ets1
in melanocyte development and to examine
potential genetic interactions between
Ets1
and melanocyte-specific transcription factors, including
Sox10
.
To this end, embryos
from crosses between
Ets1
+/-
and
Dct-LacZ
transgenic mice, in which
LacZ
expression is driven to melanocyte precursors
(
melanoblasts), were stained between embryonic days (E)11.5-15.5.
Ets1
-/-
embryos were found to have fewer melanoblasts
compared to
Ets1
+/-
and
Ets1
+/+
littermates. However, cell survival and proliferation assays suggest that
Ets1
deletion does not
result in increased melanoblast cell death or decreased proliferation at E11.5. To determine if
Ets1
interacts with
Sox10
,
Ets1
+/-
mice were crossed to
Sox10
+/LacZ
mice, in which
LacZ
was inserted in the
Sox10
locus, and the hypopigmentation phenotypes of
the progeny were compared. The areas of hypopigmentation of
Ets1
+/-
::
Sox10
+/LacZ
mice were significantly greater than the sum
of the areas of hypopigmentation of
Ets1
+/-
and
Sox10
+/LacZ
mice. The incidence of hypopigmentation in
Ets1
+/-
::
Sox10
+/LacZ
mice
was also significantly higher than that of
Ets1
+/-
and
Sox10
+/LacZ
mice. Additionally,
Sox10
expression, examined via LacZ
staining, was found to be reduced at E12.5 in 50% of
Ets1
-/-
embryos compared to
Ets1
+/+
littermates. Our results suggest
that
Ets1
is critical for melanocyte development on or before E11.5, although it does not appear to regulate melanoblast survival
or proliferation at this stage. Furthermore,
Ets1
interacts synergistically with
Sox10
and regulates its embryonic expression.
86
Essential benchmarks for use of Cre-lox mouse genetic tools. Ramkumar Sambasivan
1,2
,
Glenda Comai
1
,
Shahragim
Tajbakhsh
1
. 1)
Institut Pasteur, Stem Cells and Development, CNRS URA 2578, 25 rue du Dr. Roux, 75724 Paris Cedex 15; 2)
Institute for Stem Cell Biology and Regenerative medicine, NCBS, GKVK PO, Bellary Road, Bangalore 560065.
The vertebrate head and the associated head muscles are evolutionarily recent. We had shown earlier that the head muscle gene
regulatory network is distinct from that of the trunk, indicating that head muscle developmental program evolved independently
(
Sambasivan et al., 2009). However, all distinct upstream networks converge on the core muscle fate determining bHLH
transcription factors Myf5, Myod and Mrf4. Cell ablation studies using Myf5Cre driver lines in combination with an ubiquitous
Cre-responsive cell-death causing diphtheria toxin (DTA) allele (R26RDTA), have reported the presence of distinct muscle
progenitor populations with respect to Myf5 expression (Gensch et al., 2007; Haldar et al., 2007). The studies also reported that
the Myf5+ population is dispensable for muscle development. To carefully address the presence and role of distinct muscle