Generation of mutant mice is important in determining the
function of genes in the post-genome era. We have developed
a novel forward genetics system using the Sleeping Beauty
(SB) transposon for tagged mutagenesis in mice. SB is a synthetic
transposon reconstructed from evolutionally inactivated transposon
sequences found in the salmoid fish. The SB transposon system
consists of two elements: transposon, which acts as a mutagen,
and transposase, which recognizes and mobilizes the transposon
- known as a transposition event. Mice with transposon inserted
into functional genes can be screened rapidly and non-invasively
using GFP fluorescence. Current data indicates the usefulness
of this system for region-specific saturated mutagenesis,
which is currently impossible with other large-scale mutagenic
systems. We are currently analyzing >50 mutant lines with
transposon integration in both known and novel genes by generating
homozygotes and analyzing the resulting phenotype. To date,
we have several mutant lines which are either embryonic lethal
at various stages of development or non-lethal. Some of these
non-lethal lines display advert phenotypes such as strange
behavior pattern or infertility, while some are clearly non-essential
genes. Taken together, SB transposon system is a rapid and
efficient method for large-scale tagged mutagenesis, as well
as for saturated mutagenesis within a defined region. |
At present, the chief limitation of phenotype-based genetic
screening in mammalian systems is the diploid nature of the
genome. As it is known that cells deficient in the Bloom's
syndrome gene (Blm) show an increased rate of loss of heterozygosity,
we have developed a novel system based on this phenotype to
generate a tetracycline-regulated Blm allele in order to introduce
bi-allelic mutations across the genome in mouse embryonic
cells. Transient loss of Blm expression induces homologous
recombination not only between sister chromatids but also
between homologous chromosomes. In combination using ENU mutagenesis
and transient loss of Blm expression, we were able to generate
an ES cell library with genome-wide bi-allelic mutations.
Our results have shown that this novel system is very efficient
for identifying gene functions in ES cells. |