An effective alternate cloning strategy for unstable mouse genomic sequences.
Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector.
Lan
Michael S
MS
The Research Institute for Children, Children's Hospital, Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70118, USA. mlan@chnola-research.org
Muguira
Michelle
M
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D042822:Genomic Instability / Q000235:genetics*; D051379:Mice; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; C477059:Insm1 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors