Rat Genomics

Abstract

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 inte-gration 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. Currently, the primary means for genetically engineering rat models to study disease or gene function involves the use of pro-nuclear injection or retroviral delivery systems (1). In transgenic animals created by embryo microinjection, the site of integration of the transgene within the genome is a random event (2). Determining transgene integration sites is challenging yet with-out this information, genotyping of the rat strain is limited to identifying whether animals carry the transgene or not. The abil-ity to determine if transgene-positive animals are homozygous or heterozygous for the transgene is often not reliably possible. Additionally, when the rat strain has been created using lentiviral approaches, the ability to efficiently identify and follow segrega-tion of the multiple insertion sites that commonly occur is difficult.