A method to generate cerebellum specific knockout mice

Abstract

Cerebellum plays important role in several bodily functions such as motor coordination and balance. Genetic studies in cerebellum have gained a lot of research interest since past few decades. Cre-loxP system is one of the widely used method for genetic manipulation. Inducible Cre-loxP is the advanced and preferrable form allowing spatio-temporal modifications using cell type specific promoter and inducers such as tamoxifen. In this study, we validated cre recombinase activity in cerebellum of Gli1creERT2 mice using R26RYFP/YFP reporter mice. According to developmental stage of cerebellar granule cells, tamoxifen administration during proliferation of Granule Cells induced Gli1-mediated cre expression (YFP) in both proliferating EGL (External granule layer) cells and BG (Bergmann glia) whereas after migration of postmitotic granule cells, YFP was detected in only Bergmann glia cells. Our study suggested that different timing of tamoxifen administration could modulate cell-type specific Gli1-mediated cre-activity in cerebellum. Gαo is one of the most abundant membrane proteins in the brain despite of unknown function. Previous studies suggests that global deletion of Go protein leads to low birth and survival rate. We previously reported that Go-mediated signaling pathway regulates maturation of presynaptic parallel fibers from granule cells and climbing fibers during the cerebellar cortical development. Thus, we aimed to conditionally delete Go protein from using Gli1creERT2/+ mice. We were able to obtain enough conditional knockout mice with survival until adulthood. Here, we report efficient deletion of Go protein in cerebellum of Gli1- mediated conditional knockout mice by western blot and immunohistochemistry. Further, we performed behavior tests that are known to specify cerebellar function. Our Go conditional knockout mice showed significant deficit in motor co-ordination and learning explored by rotarod test and beam balance test. They also showed ataxic gait in gait test. Our Gli1-mediated Go conditional knockout mice is suggested to provide an efficient tool to explore more specific functions of Go protein and significantly contribute to several GPCR mediated psychiatric disorders.