Abstract
There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs). We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH) that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium). ESC germline transmission was observed in 9/11 (82%) lines using PH blastocysts, compared to 6/11 (55%) when conventional host blastocysts were used. Furthermore, less than 35% (83/240) of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137) of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the production of genetically modified animals.
Highlights
We are the product of our genes and their interaction with the environment
As Perfect Host’’ (PH) males grew towards sexual maturity it became obvious that their testes were of reduced size (,12% volume of wild type), suggesting an absence of germ cell colonization; see Figure 1 panels A and B
We describe the use of a Cre recombinase under the control of a Vasa promoter to generate embryos in which germ cells are ablated through germ cell specific expression of diphtheria toxin (DTA), whilst maintaining an environment conducive to germ cell and gamete development
Summary
Genetic engineering of the mouse enables creation of models to aid our understanding these complex interactions, providing a deeper insight of biology and genetics through the systematic modification of the mouse genome and careful characterization of the resulting animals. Recent advances in ESC line culture has improved our ability to scale the production of modified cells; see review [1], and as exemplified by The Knockout Mouse Project (KOMP) [2]. It is the stage, enhancing germline transmission of properly targeted ESC, which has lagged in development
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