Characterization & Culture of Spermatogonial Stem Cells of the Gray Short‐Tailed Opossum (Monodelphis domestica)

Abstract

Monodelphis domestica (the gray short‐tailed opossum) is a model marsupial mammal. Like all marsupials, opossums are born after brief gestation times with a complicated mix of altricial and precocial organ systems. For example, opossum forelimbs are well‐formed at birth while hind limbs are only at a paddle stage, a disparity that has been exploited to provide insights into evolution and development of the mammalian limb. The unique aspects of opossum biology also make it a good model for addressing several other questions in biomedical research (e.g. teratogenesis, regeneration, and others) and evo‐devo (e.g. middle ear formation). Opossums are also small, relatively docile, amenable to laboratory rearing, and have had their genome and many methods for their study (e.g. in situ hybridization, tissue culture, etc.) published. However, the use of opossums as a model system is currently held back by a lack of transgenic or knockout techniques. As traditional methods used in mice (i.e., pronuclear injection followed by embryo transfer) seemingly do not work in opossums, we propose to adapt spermatogonial stem cell (SSC) transplantation for use in this system. In this method, which has successfully been used in several other mammals to generate transgenic animals, SSCs are isolated, cultured, modified, and transplanted into a recipient male who is bred to generate modified offspring.As relatively little is known about marsupial SSCs, we have first characterized markers of SSC formation in rodents (e.g. Pax7, ID‐4, others) in opossums using RT‐qPCR and immunofluorescence. We found that opossum testes express several rodent SSC markers, supporting the hypothesis that opossum testes contain SSC cells. We also purified SSCs from whole testes by differential culture on several substrates. By transferring cells across a series of substrates, these methods filter out non‐SSC cells, leaving only putative SSC cells at the end. The SSC identity of differentially cultured cells from the opossum testes was confirmed using RT‐qPCR. Methods to expand, freeze, and transfect opossum SSCs are currently under development. Because SSC transplantation is often more successful when testes are cleared of endogenous SSCs, we are also optimizing busulfan treatment in opossums to evacuate endogenous SSCs from the testes. Once these methods are established, we plan to transfect opossum SSCs in culture, transfer transfected SSC cells to the evacuated testes of new males, and breed these males to generate the first transgenic opossums, and marsupials in general. This technical accomplishment will increase the utility of opossums as a model for evo‐devo and biomedical research, and thereby facilitate research into a diverse suite of evolutionary and biomedical questions.Support or Funding InformationFunding Source: NIH 1 R21 OD022988A to KESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.