Page 85 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
mutation or the genetic background. Each donor animal should be assessed; due to many parallel samples of a donor, the loss of
material remains acceptable. Several strategies are published, the gold standard might be an (resource and animal consuming)
IVF. To improve the monitoring and to save animals and resources for assessment we developed a fluorescence microscopy
based technique analyzing the membrane integrity. This parameter is significantly correlated with the outcome of paralleled IVF.
Physical and hygienic stability:
Samples should be protected against an unexpected loss, e.g. by storing parallel samples of a
line in different freezers at several locations. If not stored in the liquid phase of LN
the temperature stability must be monitored.
A powerful data management is important. Samples and receivers of revitalized embryos must be protected against infections
during handling and storage. Data of 400 cryopreserved lines are presented.
Design strategies for creation of robust TaqMan™ based assays for high-throughput genotyping of conditional knock-
out/knock-in constructs in mice. Deborah Siler
Carol Cain-Hom, Robert Schwingendorf, J. Colin Cox. Mouse Genetics
Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA.
Site-specific genomic recombinases such as Cre (recognizing loxP sites) and Flp (distinguishing frt sites) have been widely
used to generate conditional (inducible) knock-out mutations in mice and other models. The basic construct of a conditional
genetic model consists of a pair of recombination sites that flank a gene of interest. Recombinase is delivered or activated under
an inducible or tissue-specific promoter resulting in the excision of the target DNA sequence in a spatially and/or temporally
directed manner. Large-scale breeding strategies rely on highly accurate and rapid genotyping. Traditionally, genotyping has
been performed using PCR with agarose analysis or capillary electrophoresis. Here, we describe the design rationale of robust,
high-throughput TaqMan-based genotyping assays for Cre/loxP and Flp/frt-driven conditional mutations which often prove
challenging on the TaqMan platform in comparison to amplicon length-based PCR methods. Additionally, we provide strategies
for ensuring mice do not carry additional or inadvertent Cre or Flp recombinase genes which could interfere with breeding goals.
Moreover, Cre recombinase genes are often introduced with a promoter that is not expressed in a completely tissue-specific
manner, and as such, an experimentalist is highly encouraged to design to recognize all three allelic breeding states: wildtype,
conditional, and knock-out. These so-called leaky Cre promoter sets can result in the unintentional excision of target DNA
sequences and loss of the germ-line conditional construct. Finally, we describe a method for generating knock-out DNA from tail
samples using a simple
in vitro
The Sanger Mouse Genetics Project: High Throughput Recessive Lethality Screen. Antonella Galli
Jeanne Estabel,
Elizabeth Tuck, Yvette Hooks, Ed Ryder, Jacqueline K. White, Ramiro Ramirez-Solis, Tim Mohun, David Adams on behalf of
the Mouse Genetics Project.
The Sanger Institute Mouse Genetics Project (MGP) is committed to participate in the worldwide effort to develop mouse
models to understand human genetic diseases. Using the growing knockout-first conditional ready targeted embryonic stem (ES)
cell EUCOMM/KOMP resource, the MGP generates and archives over 160 lines of knockout mice per year. A standardised
battery of primary phenotyping tests is performed on all lines without any prior assumptions about gene function. The phenotypic
data and knockout lines generated provide valuable insight into novel gene functions and new mouse models of human disease.
To date, 28% of lines analysed are lethal at postnatal day 14 and a further 14% are classified as sub-viable due to reduced
homozygous viability. To explore potential defects during embryogenesis, we are collecting and assessing embryos from
heterozygous intercrosses at embryonic day 14.5 (E14.5). Any dysmorphology including growth retardation, oedema,
craniofacial, skeletal and neural tube defects are recorded and annotated. Up to now, we have analysed 162 lines and in 55%
homozygote embryos are recovered at E14.5 and a subset are further analysed using a relatively new imaging technique named
HREM (high resolution episcopic microscopy). Such technique provides highly remarkable detailed 3D models of imaged
samples, currently unachievable by alternative imaging modalities. The HREM data will be annotated and publically displayed in
the near future. Interestingly, no homozygote embryos are recovered in 45% of the lines assessed suggesting that these mutations
are embryonic lethal at an early developmental stage. Our future aim is to investigate further such mutations in order to gain new
insights into aspects of cell biology central to mouse development. Viability and anatomical abnormalities will be recorded,
annotated and analysed and available to the scientific community. Here we report a summary of recessive lethality data available
to date and examples of novel findings for a subset of interesting mutant lines.