In most species, and particularly in vertebrates, the percentage of genes absolutely required for survival, the essential genes, has not been estimated. To obtain this estimation, we used the mouse as an experimental model to carry out high-efficiency N-ethyl-N-nitrosourea (ENU) mutagenesis screens in two balancer chromosome regions, and compared our results to a third previously published screen. The number of essential genes in each region was predicted based on allele frequencies. We determined that the density of essential genes differs by up to an order of magnitude among genomic regions. This indicates that extrapolating from regional estimates to genome-wide estimates of essential genes has a huge variance. A particularly high density of essential genes on mouse Chromosome 11 coincides with a high degree of regional linkage conservation, providing a possible causal explanation for the density variation. This is the first demonstration of regional variation in essential gene density in the mouse genome.Citation: Hentges KE, Pollock DD, Liu B, Justice MJ (2007) Regional variation in the density of essential genes in mice. PLoS Genet 3(5): e72.

The telencephalic vesicles form in the mouse embryo by the expansion of precursor regions in the anterior neural tube. Once the vesicles have formed, discrete dorsal and ventral territories can be recognized that later give rise to cortical and subcortical structures, respectively. To investigate the mechanisms that regulate the expansion and regionalization of the telencephalon, we have carried out a screen to identify recessive mutations that disrupt these events. We isolated a mouse mutant in which an early and critical step in development of the telencephalic vesicles is disrupted. Telencephalic primordia are present in flat-top embryos but they fail to progress to form the telencephalic vesicles. An increased rate of proliferation in the forebrain neurectoderm that accompanies telencephalic expansion in wild-type embryos fails to occur in flat-top embryos. Regionalization events that would normally take place during expansion of the primordia also fail to occur. Thus the phenotype of the flat-top mouse reveals that outgrowth of the telencephalic vesicles and their regionalization are coupled processes.

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

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