Precis on the ΦX174 (Malling Mouse)
- ΦX174 Maps (pdf format)
- ΦX174 Sequence
- ΦX174 database first pass (Excel format)
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Bacteriophage |
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 |
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 |
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 |
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™ animal. |
