Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats

Toshihiro Kobayashi,Hiromitsu Nakauchi,Makoto Sanbo,Masumi Hirabayashi,Yasuhiro Kazuki,Fumika Yoshida,Shinichi Hochi,Teppei Goto,Naoyo Kajitani,M Azim Surani,Kanako Kazuki,Mami Oikawa,Naoko Niizeki,Reiko Terada

doi:10.1038/s41467-021-21557-x

Abstract

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.

Highlights

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies
While recent progress in genome editing technologies such as CRISPR/Cas[9] in zygotes have enabled the generation of desired animals rapidly and efficiently[1,2], PSCs are useful for precise genetic modifications[3]
For a suitable host blastocyst lacking the germline, we focused on Prdm[14], a key transcriptional regulator essential for the specification of primordial germ cells (PGCs), the founder cells for gametes

Summary

Result

Blastocyst complementation using Prdm[14] KO rats enables efficient germline transmission of allogenic embryonic stem cells (ESCs). All the generation rats obtained after crossing with wild-type rats exhibited tdTomato fluorescence and dark skin (male: 85/85 [100%], female (31/31 [100%], Fig. 1h, Supplementary Fig. 1a, b, Table 1, Supplementary Table 1), suggesting complete germline transmission of donor rat ESCs without any host-derived germ cells. All the germline in testes and ovaries of Prdm[14] KO rats are composed of tdTomato expressing cells (Fig. 2b), confirming our previous result (Fig. 1), that the germline is complemented by donor Pax2/Pax[8] KO rat ESCs. we crossed male and female chimeras with Pax2/Pax[8] KO gametes to obtain Pax2/ Pax[8] double KO rats. In chimeras obtained from Prdm[14] KO rat blastocysts, we could observe seminiferous tubules filled with EGFP-positive cells in the adult testes (Fig. 2e). We demonstrated that interspecific blastocyst complementation in the germline can yield functional mouse spermatids in rats

Discussion

Methods