title: An effective alternate cloning strategy for unstable mouse genomic sequences.
abstract: 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.
authors.affiliation: 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
authors.forename: Michael S
authors.initials: MS
authors.lastname: Lan
authors.forename: Michelle
authors.initials: M
authors.lastname: Muguira
mesh_terms: 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*
chemical_list: D004268:DNA-Binding Proteins; C477059:Insm1 protein, mouse; D012097:Repressor Proteins; D014157:Transcription Factors