Page 83 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
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compatibility with other episomal elements; and 4) high cloning efficiency with no multimerization. To test our approach, we
used the mini- recombineering system to insert homology arms into our retrieving vector, and then efficiently subcloned a 65 kb
BAC region into our retrieving vector without a significant percentage of aberrant recombinants. We believe that the combined
benefits of our new approaches may decrease costs and simplify mouse conditional mutagenesis by eliminating the need for using
selection cassettes and the Gateway technology.
139
Crucial elements of contemporary genotyping assay design criteria and screening procedures in the context of a very
high-throughput genetic analysis laboratory. Colin Cox
,
Deborah Siler, Carol Cain-Hom, Ryan Pabalate, Rhonda Wiler.
Mouse Genetics, Genentech, South San Francisco, CA.
The ability to rapidly genotype genetic mouse models with an extremely high degree of accuracy is a critical function of a
genetic analysis laboratory or animal research facility. We present three arenas which are topically important for current genetic
screening and validation needs. First, we present a resilient methodology for design of TaqMan genetic screening reactions and
show how this results in a greater than 50% reduction of failed reactions in comparison with PCR. Traditional screening methods
like PCR are no longer considered high-throughput and moreover, PCR itself is not as sensitive or selective as TaqMan.
However, while it may be technically straightforward to develop TaqMan assays for simple knock-out, knock-in, and transgene
models, it can be difficult to design robust assays capable of resolutely interrogating the three allelic states of a conditional
knock-out or knock-in model and we detail how to overcome this challenge. Second, we show how it is essential to monitor for
all three allelic states in order to prevent the inadvertent germ-line loss of a conditional gene owning to leaky expression of what
should be specific promoter-driven of a recombinase (Cre, Flp, and Dre). In order to easily validate tissue-specific knock-out
detection from simple tail or ear clippings, we have developed a simple in vitro assay capable of generating (and detecting) the
null allele. Third, we demonstrate the need for implementation of quality assurance validation procedures, both for in-house
model creation as well as for acquisition of models from collaborators. Models can be incorrectly created, especially mature
models made over a decade ago and/or models using older transgenic creation technology such as simple cloning and restriction-
site based assemblage. Quality assurance validation can be especially important for purchased models which may harbor
unintentional genes such as a hidden recombinase or genetic selection marker. Finally, we describe other quality and validation
opportunities such as single-plex corroboration, DNA sequence analysis verification, matching primer and probe Tms during the
design process, etc. In the limit, we show how all these functions serve to design and execute assays that fully meet the
investigational needs of a research group in the context of a very high-throughput genetic analysis laboratory performing 750,000
genotyping reactions annually across 1,000 distinct mouse models.
140
Whole Body Inducible Knockouts in Mice Using a Novel Tet-Inducible Cre-LoxP System. Joseph E Dinchuk
,
Fanny
Myers, Amy Weiss, Christopher Miller. Functional Genomics, Bristol-Myers Squibb R & D, Princeton, NJ.
There are currently no genetic systems available that allow for a whole body adult robust induction of gene expression that
includes the CNS. We devised and describe genetically engineered mice, based upon the tetracycline inducible system, that
allows for non-leaky, whole-body inducible and reversible gene expression in the mouse and potentially other mammals. Our
first application of this system is the production of a mouse with non-leaky and whole-body induction of Cre recombinase. The
availability of such a mouse and adaptation of the Cre-Lox system to it allows the production of mice with the capability of a
whole body induction of adult KOs in all organs including those in the CNS. The first component of this system consists of a
genetic element that ubiquitously co-expresses a reverse tetracycline transactivator and forward tetracycline transcriptional
inhibitor from the Rosa26 locus. This unique combination of elements assures that the responder gene should be both non-leaky
and cleanly inducible upon application of tetracycline or tetracycline derivatives to such mice. The second component of this
system is a tet-operator genetic element that co-expresses both a Cre recombinase and a reverse tetracycline transactivator from a
favorable locus on the X-chromosome. This second system essentially adds a turbo-drive to the induction system in order to
allow for high levels of the target gene expression (in this case Cre recombinase). When we combined mice containing these 2
genetic elements with a floxed transgene known to express highly in the CNS (and elsewhere) and fed the mice doxycycline, we
were able to demonstrate virtually a complete knockout of the gene in kidney tissue along with a 95% knockout of the same gene
in the cerebral cortex. We are currently repeating these experiments using different doses of doxycycline and targeting different
floxed genes. We expect this system to be a useful for adult induction of gene expression and especially useful for gene knockout
systems when the gene that is regulated is Cre recombinase.
141
A method for the conversion of PCR-based fragment analysis genotyping assays to 5’ nuclease assay (TaqMan™)
platform with validation. Ryan Pabalate
,
Robert Schwingendorf, Deborah Siler, Gregg Sy, Colin Cox. Genentech, Inc, South
San Francisco, CA.
The ability to quickly and robustly genotype genetic mouse models is crucial in an animal care facility in order to rapidly
segregate genetically distinct pups in a given litter. Currently, many facilities utilize PCR for genetic screening by either
employing agarose gels or capillary electrophoresis-based PCR amplicon detection. While functional, this method is inferior in
comparison to other technologies such as the 5 nuclease assay (TaqMan). First, automated technologies like capillary
electrophoresis machines may be capable of only processing 96 samples per hour. In comparison, TaqMan instrumentation can