Page 82 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
genetic background. In contrast to 129 mouse ES cell lines, the use of a standard inbred C57BL/6 mouse background reduces
variation in experimental measurements and permits comparison of results to a wealth of published data. The C57BL/6 Bruce4
ES cell line determined to be genetically instable during a series of gene-targeting projects. Gene-targeted ES cell clones were
expanded, characterized by chromosome counting, and tested against a functional definition of euploidy to assess the genetic
stability. For each ES cell clone, 20 chromosome spread were counted. If twelve or more spreads contained the normal
chromosome number for mouse (40 chromosomes) the clone was scored as euploid. The functional definition of euploidy was
predicated on the likelihood of the formation of germline ES cell-mouse chimeras. The results showed that only 35% of correctly
targeted ES cell clones were euploid and suitable for the preparation of chimeras. In an attempt to isolate a robust C57BL/6 ES
cell line for gene targeting, a series of Bruce4 sublines were developed and characterized by chromosome counting and formation
of germline chimeras. One subline, Bruce4.G9, contained 90% euploid chromosome spreads and formed germline ES cell-mouse
chimeras. A series of gene targeting experiments were then conducted to compare Bruce4 and Bruce4.G9 subline ES cells in
parallel. Consistent with prior observations, only 26% of gene-targeted Bruce4 clones were euploid. However, 100% of gene-
targeted Bruce4.G9 clones were euploid. A sample of euploid gene-targeted clones was used to prepare ES cell-mouse chimeras
and tested for germline transmission. Bruce4 clones were less efficient (40% of clones were germline) than Bruce4.G9 clones
of clones were germline). Genet targeting efficiency in the Bruce4.G9 subline is superior compared to the parental Bruce4
ES cell line. A genetically stable ES cell subline with high germline potential was established from a parental ES cell line
characterized by genetic instability and weak germline potential. These results indicate that mouse ES cell lines with suboptimal
performance can be rescued by establishing sublines with robust gene-targeting characteristics.
Forward genetics identifies Edf1 as a novel regulator of epidermal development and stem cell quiescence. Scott D
Sunjin Lee-Wölfel
Yong Kong
. 1)
Genetics, Yale University, New Haven, CT; 2) Molecular Biophysics and
Biochemistry, New Haven, CT; 3) W.M. Keck Foundation Biotechnology Resource Laboratory, New Haven, CT.
The outermost layer of the skin, the epidermis, plays a key role in animal survival by acting as a barrier to prevent infection and
desiccation. Stem cells in the interfollicular epidermis undergo a series of cell fate choices during the differentiation program to
form a stratified epidermis. The appropriate balance between proliferation and differentiation is crucial for epidermis function,
and alterations in this process can cause human diseases, such as psoriasis and skin cancer. However, the factors that regulate cell
fate choices of stem cells in the epidermis are not well understood. To identify new mediators involved in these processes, we
performed a forward genetics screen in mice and identified a novel regulator of skin development, the
Epidermal differentiation
factor 1
gene. Mice carrying a homozygous mutation in
develop a hyperproliferative, poorly differentiated
epidermis. We have shown that
function is essential to curb stem cell proliferation and for normal differentiation of their
progeny. We further demonstrate that
and the cell cycle regulator
Sfn; 14-3-3
act together to regulate keratinocyte
differentiation and epidermal barrier formation. The transcription factor p63 is a master regulator of epidermal development and
strongly expressed in the stem cell compartment.
mutants, however, exhibit increased levels of p63 throughout the
epidermis and reduction of p63 dosage in
mutants rescues many aspects of the phenotype, indicating that Edf1 modulates
p63 levels. Together, our findings identify Edf1 as a novel regulator of epidermal stem cell proliferation and differentiation that
regulates p63 expression and acts with Sfn to balance these processes.
Does gene targeting for the generation of mouse conditional alleles still require selection markers and Gateway
technology? Luca Brunelli
George Lezin
Yasuhiro Kosaka
H. Joseph Yost
Michael R. Kuehn
. 1)
The University of Utah,
Salt Lake City, UT; 2) Laboratory of Protein Dynamics and Signaling, NCI-Frederick, Frederick, MD.
Targeting vector construction for mouse conditional mutagenesis currently involves inserting the selection cassettes necessary
for isolating various intermediate recombinant constructs. The Gateway technology is also commonly required at multiple
intermediary steps and/or for the final assembly of the targeting vector. As these approaches remain fairly complex, time
consuming and expensive, we investigated whether recombinants could be isolated without using selection markers, as well as
whether we could develop a low-cost in vivo cloning method to replace the Gateway technology. First, we determined whether
the founder principle, a population genetics concept, could be used to isolate markerless recombinants. After inserting the coding
sequences of the fluorescent proteins mStrawberry or eYFP into a mouse Nodal bacterial artificial chromosome (BAC), we
isolated markerless and seamless mStrawberry and eYFP Nodal BAC recombinants using the founder principle and PCR
screening in liquid cultures. Derived transgenic mice expressed mStrawberry and eYFP in a pattern consistent with Nodal.
Secondly, using gap repair cloning, a recombineering application using DNA repair to clone DNA into a retrieving vector, we
developed a simple and highly efficient in vivo cloning system that is low-cost because it does not require enzymes. This system
is based on a new P1 phage-based retrieving vector constructed in our laboratory and which contains four key structural and
functional improvements: 1) similar cloning capacity to BACs (~300-kb); 2) copy number modulability; 3) expected