We have previously demonstrated that the tetraspanin CD9 is necessary for membrane fusion between sperm and oocyte during fertilization. While knockout mice for CD9 are viable, CD9-/- females are sterile due to the inability of their oocytes to fuse with sperm. While CD9 is not essential for subsequent development, a role in embryonic stem (ES) cell self-renewal was hypothesised on the basis of two observations: CD9 is highly expressed in murine and human ES cells and the CD9-blocking antibody inhibits mouse ES cell colony formation and survival. To investigate whether CD9 has a direct effect on ES cells, we generated and characterised several CD9 knockout murine ES cell lines. These CD9-/- ES cell lines exhibited equivalent morphology and growth properties to wild-type ES cells. Furthermore, the CD9-/- ES cell lines also displayed similar expression of pluripotency factors Oct3/4, Sox2 and Nanog. CD9-/- ES cells were found to be pluripotent in vivo, as their cells injected into immunocompromised mice gave rise to teratomas consisting of tissues representative of all three germ layers. Additionally several high contribution mouse chimeras were generated by blastocyst injection with several CD9-/- ES cell lines. Taken together, our results reveal that CD9 is dispensable for mouse ES cell self-renewal and pluripotency. The generation of CD9-/- ES cells should prove to be a useful tool with which to study the function of this protein and a range of other associated cellular processes.
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