Page 47 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
Characterization of haploid embryonic stem cells from mouse embryos. Anton Wutz
Martin Leeb. Wellcome Trust Centre
for Stem Cell Research, Cambridge, Cambridgeshire, United Kingdom.
Haploid embryonic stem cells (ESCs) have recently been derived from mouse parthenogenetic embryos and applied for genetic
screens in culture. These cells maintain key properties of mouse ESCs and might overcome existing limitations in developmental
genetic approaches in mice. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and
in vitro has been demonstrated and is paralleled with the gain of a diploid karyotype. It has remained unclear if haploid ESCs can
contribute to the germline. Here we show that parthenogenetic haploid ESCs have robust germline competence enabling the
production of transgenic mouse strains from genetically modified haploid ESCs. We further show that differentiation of haploid
ESCs in the embryo correlates with an efficient gain of a diploid karyotype and that diploidization is likely the result of
endoreduplication and not cell fusion. In contrast to the embryo a haploid karyotype is maintained under certain conditions
during in vitro differentiation.
MFA: A Novel Method For The Generation Of “All-In-One” Null And Conditional Alleles.
Christopher Schoenherr,
Evangelos Pefanis, Peter Lengyel, Darshi Persaud, YuHong Zhang, Ronald Deckelbaum, Julie Kalter, Dimitrios Skokos, Peter
Yang, Andrew J Murphy,
Aris N. Economides
Regeneron Pharmaceuticals, Tarrytown, NY.
The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. We
developed a method - Conditional by Inversion (COIN) - that utilizes an exon-splitting intron and an invertible genetrap-like
module (COIN module) to create null alleles upon Cre-mediated inversion. While COINs provide unprecedented flexibility in
engineering conditional alleles, we have extended COINs by generating a new Multifunctional Allele (MFA), which utilizes a
single gene-targeting step and three site-specific recombination systems, to generate four distinct allelic states: (1) Null with
reporter (plus drug selection cassette): MFAs start by replacing the gene of interest with a modified version of the gene that is
inactivated by both inversion of a critical part of the coding sequence and simultaneous insertion of a reporter gene and selection
cassette. This is accomplished by gene targeting. (2) Null with reporter (minus drug selection cassette): MFAs allow the option of
subsequent deletion of both the selection cassette and the inverted gene segment using site-specific recombination (Dre). This
gives the best reporter gene expression while retaining gene inactivation. (3) COIN-based conditional-null: MFAs can be
converted to a phenotypically wild-type, yet conditional allele, via removal of the selection cassette and reporter and
simultaneous re-inversion of the critical part of the gene through the action of a second site-specific recombinase (FLP). This step
also acts as a built-in complementation test that addresses the possibility of artefacts introduced during the targeting process. (4)
Recombinase-induced null: MFAs allow re-inactivation of the conditional allele created in step 3, by re-inversion of the critical
gene segment using a third site-specific recombinase (Cre), and concomitant induction of a second reporter (that embedded in the
COIN module). Using
as a test locus, we have determined that MFA works robustly to generate all four functionalities.
The results obtained with
are being extended to a set of genes with well-characterized knockout alleles, such as
in order to provide a stringent comparison with their MFA counterparts. Lastly, given that MFAs rely on standard genetic
engineering, gene targeting, and site-specific recombinase systems, they could be easily adopted by laboratories skilled in
generating genetically modified mice without any changes in current workflow.
Efficient generation of a conditional knock-out allele in mice by zinc finger nuclease-mediated gene targeting.
Jeremy Burton
Tuija Alcantar
Jinjie Li
Robert Schwingendorf
Tim Soukup
Merone Roose-Girma
J. Colin
Xin Rairdan
Weilan Ye
Soren Warming
. 1)
Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South
San Francisco, CA 94080; 2) Department of Mouse Genetics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080.
The use of sequence-specific zinc finger DNA-binding domains fused to an endonuclease domain (zinc finger nucleases, or
ZFNs) has enabled efficient and direct generation of both gene knock-out and knock-in alleles by microinjection into fertilized
eggs from either mice or rats. While knock-out alleles result from error-prone repair of a ZFN-mediated double-strand break
DSB), knock-in alleles are obtained by co-microinjection of ZFN mRNA and a donor plasmid that serves as a template for
repair. However, existing methods are not suitable for generating conditional knock-out alleles due to extensive homology
between host and donor sequences and the presence of the ZFN target site in the donor plasmid. We now describe the successful
generation of mice carrying a conditional knock-out allele of the Lrp5 gene using ZFN technology. This was achieved by
microinjection of mRNA encoding a highly efficient ZFN pair along with a donor plasmid containing a loxP-flanked (floxed)
exon sequence with a number of silent mutations to reduce unintended cross-over and ZFN cleavage in the region between the
two loxP sites. When using a donor plasmid with a high degree of homology to the target exon, we instead obtained mice with
alleles missing either one or both loxP sites. This conditional knock-out strategy and the results have important ramifications not
only for mouse genome engineering using ZFN technology, but also for genetic engineering of other mammalian species.