A restriction enzyme-PCR-based technique to determine transgene insertion sites.
Currently, most genetically engineered rat strains are created by methods that involve random integration of transgenes into the genome. The ability to identify the chromosomal location of the transgene insertion site enables the development of efficient genotyping assays, allows segregation of multiple transgene integration sites to be followed while breeding, and facilitates characterization of possible positional effects on phenotype. Here we describe a method for determining the chromosomal location of transgene insertion that combines restriction endonuclease enzyme digest with subsequent rounds of PCR amplification to produce amplicons representing the chromosomal regions flanking the integrated transgene. This method provides a reliable means for determining the exact location of insertion of transgenes within the genome.
Bryda
Elizabeth C
EC
Research Animal Diagnostic Laboratory, University of Missouri, Columbia, MO, USA.
Bauer
Beth A
BA
D000818:Animals; D001483:Base Sequence; D002875:Chromosomes / Q000235:genetics; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D017422:Sequence Analysis, DNA / Q000379:methods; D019076:Transgenes
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D004247:DNA; D004262:DNA Restriction Enzymes