Transgenic and In Vivo Mutagenesis
Precis on the ΦX174
(Malling Mouse)
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Bacteriophage
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The bacteriophage ΦX174 transgenic
mouse, also referred to as the Malling mouse, after its founder, Dr.
Heinrich V. Malling, contains approximately 50 tandem copies of the
E. coli bacteriophage ΦX174 in an as yet unknown chromosomal
location in a C57/Bl6 background. ΦX174 is one of the smallest
bacteriophages known, containing only 5386 nucleotides in a
single-stranded DNA genome, and 10 genes, A through K. The
bacteriophage capsid is a symmetrical icosahedral structure (no
tail). The viral genome is excised from the mouse chromosome by
digestion with the restriction enzyme, PstI, circularized and
ligated to form the circular, double-stranded replicative form DNA,
and introduced into the bacterial recovery cell directly by
electroporation.
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Reversion and
Mutation Assays
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The ΦX174 genome integrated into the
mouse is mutant at the am3 locus in gene E and forms the basis for a
reversion assay, detecting base pair substitution at an A:T base
pair. A forward mutational assay has also been developed that
requires a gain of function to compensate for an E. coli mutation in
the rep gene. About 90% of isolated mutants are found in gene A;
however, the forward selection assay also identifies mutations in
genes F and C, because the method of compensation involves a complex
containing these proteins. Therefore, this assay also detects only
base pair substitution at 55 currently known target sites. All six
types of base pair substitution are recovered by this forward assay.
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Single Burst Analysis
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The spontaneous mutant frequency of
ΦX174 is approximately 2 x 10-5 and is about 10 times
higher than the mutation frequency of this transgene measured in
mouse cells. The lower mutation frequency can be measured by use of
a method called "single burst analysis" in which the electroporated
culture of bacterial cells is divided into numerous aliquots
(typically 96) before replication of the bacteriophage and lysis of
the bacterial cell. When each aliquot is plated on a separate agar
plate, only those mutations fixed in the mouse cell produce a large
burst of progeny virus on the same plate (at least >55 plaques).
This technique allows the elimination of mutants fixed in the
bacterial cell from damage caused in the mouse cell if two cell
replications are needed to fix the damage into a mutation.
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Mutagens Tested
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To date (2007), single burst analysis of
the forward mutational assay has been applied to two mutagens, UVB
and ENU. UVB mutagenesis of a ΦX174 mouse embryonic cell line has an
induced mutant frequency per nucleotide equivalent to that of cII,
but a spontaneous mutant frequency an order of magnitude lower; in
mouse spleen, the ΦX174 forward assay has a similar mutagenic
response to ENU as that of the lacI transgene of the Big Blue(TM)
animal.
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