Format

Send to

Choose Destination
Stem Cell Reports. 2015 Jul 14;5(1):75-82. doi: 10.1016/j.stemcr.2015.05.011. Epub 2015 Jun 18.

Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9.

Author information

1
Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan. Electronic address: tsato@yokohama-cu.ac.jp.
2
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
3
Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
4
Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan.
5
RIKEN, Bioresource Center, Ibaraki 305-0074, Japan.
6
Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan; Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address: ogawa@yokohama-cu.ac.jp.

Abstract

Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

PMID:
26095606
PMCID:
PMC4618438
DOI:
10.1016/j.stemcr.2015.05.011
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center