Dre - Cre Sequential Recombination Provides New Tools for Retinal Ganglion Cell Labeling and Manipulation in Mice

BACKGROUND: Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations. METHODS AND FINDINGS: We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3a(CKOCre) and Brn3b(CKOCre). When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3a(CKOCre) or Brn3b(CKOCre) is removed and replaced with a Cre recombinase, generating Brn3a(Cre) and Brn3b(Cre) knock-in alleles. Surprisingly both Brn3a(Cre) and Brn3b(Cre) knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3a(Cre) and Brn3b(Cre) alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs. CONCLUSIONS: Dre recombinase effectively recombines the Brn3a(CKOCre) and Brn3b(CKOCre) alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3a(Cre) and Brn3b(Cre) alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.