Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines:Prevalence of Germline Recombination and Influencing Factors

Luo et al. report variable rates of germline recombination in commonly used mouse Cre driver lines, influenced by sex of Cre-carrying parents and target loci. Guidelines are provided to optimize cell-type-specific recombination in genetically targeted organisms expressing site-specific recombinases

Calcium release at fertilization: Artificially mimicking the oocyte's response to sperm

The mechanism of sperm-induced calcium release has been the subject of many studies since the development in the late 1950s of in vitro culture systems that support mammalian fertilization. Despite efforts to elucidate the nature of the signal from the sperm that triggers both the early and late events of oocyte activation, the precise mechanism remains unresolved. Now, with the advent of somatic nuclear transfer technologies, the need to better understand this unique process has been recognised. Nuclear transfer embryos must be induced to commence development artificially because the activating signal from the sperm is absent. The primary activating stimulus is a large increase in the concentration of intracellular-free calcium and numerous physical and chemical treatments have been found to induce calcium changes that initiate the events of oocyte activation. Although live cloned offspring have been produced in a number of species, the overall efficiencies of the nuclear transfer procedures described thus far are unacceptably low and phenotypic anomalies are common. With the aim of improving these efficiencies, researchers are developing artificial activation treatments which induce oocyte responses that mimic those induced by fertilizing sperm. One strategy is to replicate the pattern of calcium change more closely. Another strategy is to couple an activating stimulus with treatments that inhibit maturation (or M-phase) promoting factor (MPF) activity, which regulates meiotic progression in oocytes. This paper reviews what is understood of calcium release at fertilization and describes the treatments that have been used to induce oocyte activation artificially in parthenogenetic and nuclear transfer studies. The relative effectiveness of the strategies employed to mimic the oocyte's response to sperm are discussed.Christopher G. Grupen, Mark B. Nottle and Hiroshi Nagashim