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Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies.

In the recent years, sequence-specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionzed the fields of animal genome editing and transgenesis. However, these new techniques require microinjection to deliver nucleic acids into embryos to generate gene-modified animals. Microinjection is a delicate procedure that requires sophisticated equipment and highly trained and experienced technicians. Though over a dozen alternate approaches for nucleic acid delivery into embryos were attempted during the pre-CRISPR era, none of them became routinely used as microinjection. The addition of CRISPR/Cas9 to the genome editing toolbox has propelled the search for novel delivery approaches that can obviate the need for microinjection. Indeed, some groups have recently developed electroporation-based methods that have the potential to radically change animal transgenesis. This review provides an overview of the old and new delivery methods, and discusses various strategies that were attempted during the last three decades. In addition, several of the methods are re-evaluated with respect to their suitability to deliver genome editing components, particularly CRISPR/Cas9, to embryos. Reviewers: Drs. Eugene Koonin and Haruhiko Siomi.

Background The mouse has more than 30 Major histocompatibility complex (Mhc) class Ib genes, most of which exist in the H2 region of chromosome 17 in distinct gene clusters. Although recent progress in Mhc research has revealed the unique roles of several Mhc class Ib genes in the immune and non-immune systems, the functions of many class Ib genes have still to be elucidated. To better understand the roles of class Ib molecules, we have characterized their gene duplication, organization and expression patterns within the H2 region of the mouse strain C57BL/6. Results The genomic organization of the H2-Q, -T and -M regions was analyzed and 21 transcribed Mhc class Ib genes were identified within these regions. Dot-plot and phylogenetic analyses implied that the genes were generated by monogenic and/or multigenic duplicated events. To investigate the adult tissue, embryonic and placental expressions of these genes, we performed RT-PCR gene expression profiling using gene-specific primers. Both tissue-wide and tissue-specific gene expression patterns were obtained that suggest that the variations in the gene expression may depend on the genomic location of the duplicated genes as well as locus specific mechanisms. The genes located in the H2-T region at the centromeric end of the cluster were expressed more widely than those at the telomeric end, which showed tissue-restricted expression in spite of nucleotide sequence similarities among gene paralogs. Conclusion Duplicated Mhc class Ib genes located in the H2-Q, -T and -M regions are differentially expressed in a variety of developing and adult tissues. Our findings form the basis for further functional validation studies of the Mhc class Ib gene expression profiles in specific tissues, such as the brain. The duplicated gene expression results in combination with the genome analysis suggest the possibility of long-range regulation of H2-T gene expression and/or important, but as yet unidentified nucleotide changes in the promoter or enhancer regions of the genes. Since the Mhc genomic region has diversified among mouse strains, it should be a useful model region for comparative analyses of the relationships between duplicated gene organization, evolution and the regulation of expression patterns.