Abstract
Cre-LoxP conditional knockout animals have become a prominent tool to understand gene function in discrete cell-types and neural circuits. However, this technology has significant limitations including off target cre-dependent recombination. The Rgs9cre strain has been used to generate a conditional knockout in striatal medium spiny neurons, but, as presented in the current study, off target recombination in the germline results in nonconditional deletion of LoxP alleles. Using a Rem2 conditional allele, germline deletion (GD) was observed in a sex dependent manner. When Cre and LoxP alleles were co-inherited from the female parent, 27 of 29 LoxP alleles were recombined, but when co-inherited from the male parent, 5 of 36 LoxP alleles were recombined. Rem2 expression measured by RT-qPCR confirmed nonconditional recombination in extrastriatal nuclei. Cre-LoxP is a powerful technique to modify genomic DNA (gDNA), however careful characterization of these mice is required to confirm control of conditional recombination.
Highlights
Conditional knockouts using Cre-LoxP technology have been instrumental in dissecting the function of proteins in neurophysiology by allowing gene deletion or gene expression with temporal, tissue and cell-type specificity
If recombined alleles detected in ear biopsies is due to germline recombination, mRNA should be present in the gonads of Rgs9cre mice
The current report demonstrates germline recombination of Rem2 conditional alleles when breeding with the Rgs9cre driver line, which occurred at higher frequency when the Cre allele was present on the female parent
Summary
Conditional knockouts using Cre-LoxP technology have been instrumental in dissecting the function of proteins in neurophysiology by allowing gene deletion or gene expression with temporal, tissue and cell-type specificity. Mutant mice containing a floxed (i.e., flanked by LoxP) gene of interest are bred with mice expressing Cre driven by a tissue or cell-type specific promoter These Cre-driver lines are generated by identifying a gene with advantageous expression patterns and inserting the Cre coding sequence into either the endogenous gene locus or a bacterial artificial chromosome (BAC) harboring the gene promoter of interest (the BAC is subsequently introduced into the host genome; Parkitna et al, 2009). The current report describes germline recombination in the Rgs9cre line (Dang et al, 2006), which has been widely used to manipulate medium spiny neurons in the striatum
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