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Targeted gene alteration in Caenorhabditis
elegans by gene conversion Peter L Barrett, John
T Fleming  Verena Göbel Nat Genet. 2004 Oct
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• Present methods for isolating mutations in
  specific gene in
• C. elegans
• using transposon insertions at least 8
  distinct transposons have been identified in C.
  elegans mutator strains with 400 times higher
  efficiency than wild type
• using chemical- or radiation-based mutagenesis
• Both methods use PCR for the gene-specific
  detection of deletions the location and size of
  a deletion can be controlled only imprecisely by
  the selection of the primers many worms have to
  be tested
• Homologous recombination occur only rarely in C.
  elegans - is not yet used routinely.

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Strategy for targeted gene alteration by gene
conversion a way to create an engineered
deletion in the gene
Transposon excision ? double-stranded DNA (dsDNA)
breaks, which are thought to be repaired in a
template-directed manner by means of the sister
strand A transgene can also act as a template
for repair after the excision of a Tc1 transposon
in C. elegans A transgene containing an
engineered deletion of a specific size in the
genomic DNA corresponding to the area of the Tc1
insertion site can be used as template for repair
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Isolation of targeted alleles
2 different genes containing Tc1
transposons tkr-1 conversion plasmid
contained a 0.85 kb deletion frm-3 conversion
plasmid contains a 1.5 kb deletion Generation
of transgenic lines containing the respective Tc1
alleles and conversion plasmids rol-6 and
sur-5GFP as markers. tkr-1 was tested in mut-2
mutator background frm-3 was tested in mut-2 and
mut-7 backgrounds 5-10 parent worms ? population
of 500 1,000 worms Isolation of DNA from
about 1/3 of population ? Using gene-specific
primer pairs to amplify only the gene-converted
product, not the transgene
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tkr-1
Frequency of gene conversion pilot study with 16
populations tested 2 positives ? 2 in 5,333
(much higher than 1 in 100,000 previously
reported for point mutations) Confirmation of
deletion by sequencing, Southern blots and
negative PCR results with primers matching
sequences inside the deletion Absence of
transgene loss of roller or GFP and inability to
amplify transgene vector sequences from strains
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frm-3
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Frequencies in different genetic backgrounds

• Comparison of 3 independently derived transgenic
  mut-7 strains carrying the frm-3 Tc1 and the
  conversion plasmid
• Strains are different in viability
• transgene copy number and
• transmission rate
• 45 populations of each strain were assayed
• Similarly high results of 1-3 out of 45
  populations
• health of the strains
• the resulting number of generations needed to
  populate the plate and the properties of the
  array
• are not essential for obtaining gene conversion
• Frequency in mut-2 background was 3 times higher
  than in mut-7
• Same frequency of tkr-1 (0.85 kb deletion) and
  frm-3 (1.5 kb deletion) in mut-2 background

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Generation insertion-replacement alleles
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Advantages of this method

• High frequencies
• No screening of large numbers of worms one to
  three orders of magnitude lower than in
  previous screening methods
• generating custom alleles
• GFP insertions allowing examination of gene
  expression in single copy number, in its native
  genomic milieu and under physiological conditions