16 Spermatogonial stem cells (SSCs) are derived from postnatal quiescent progenitor cells and have the unique ability to self renew or divide into more differentiated progeny (Potter and DeFalco 2017). The capability for self renewal and differentiation in adult mammalian enables SSCs to maintain spermatogenesis (Azizi et al. 2017). SSCs are unique stem cells that transmit genetic information to offspring and can be manipulated to produce transgenic animals (Wang et al. 2014). Aim of the present study was to define the optimal transfection conditions of SSCs in early and late ovine SSC colony formation stages in culture. Liposomes are made up of cationic lipids, which can interact with the negatively charged nucleic acid molecules and form complexes coating the nucleic acid inside. The positive outer surface of the complex can associate with the negatively charged cell membrane, allowing the internalization of the nucleic acid (Niu and Liang 2008). Liposome-mediated transfection (lipofection) is a simple and powerful technique for DNA transfer into cultured cells (Kalina et al. 2003). Turbofect is a cationic polymer and can be used in the presence or absence of serum (Oba and Tanaka 2012). Indian Journal of Animal Sciences 87 (8): 944–946, August 2017/Article Transfection of early and late stage sheep spermatogonial stem cells in culture F JASOUR 1 , M ZANDI 2 , K HOSEINI PAJOOH 3 , M R SANJABI 4 and H OFOGHI 5 Iranian Research Organization for Science and Technology, Tehran 33535111 Iran Received: 25 May 2016; Accepted: 15 May 2017 ABSTRACT The genetic manipulation of spermatogonial stem cells (SSCs) can be used as an alternative to somatic cell nuclear transfer method for the production of transgenic animals. SSCs are now in vitro cultured and transplanted in sheep, however, there are no known protocol for DNA transfection of sheep SSCs. The aim of present study was to define the optimal transfection conditions of spermatogonial stem cells (SSCs) in early and late ovine SSC colony formation stages in culture. SSCs were isolated from the slaughterhouse ram testis tissue using a two-step enzymatic digestion process. Results showed that, 2 μl of DNA with 0.5 μg of Lipofectamin or Turbofect were able to transfect SSC colonies at late stage. Since the colonies of SSCs were not in logarithmic growth phase, around 15% of colonies were transfected and no significant difference between Lipofectamin or Turbofect was observed. However, more cells were transfected on early stages of SSC colony formation (7 th days), especially when Turbofect was used (around 40 and 45% for Lipofectamin and Turbofect, respectively). Although the early stages SSCs were more suitable for transfection, but the formation of colonies were impaired on transfected cells. Key words: Lipofectamine, Sheep, Spermatogonial stem cells, Transfection, Turbofect MATERIALS AND METHODS Collection of testes and isolation of SSCs: Ram lambs testes collected from slaughterhouse immediately after slaughter were washed 3–4 times with normal saline solution (32–37ºC), containing 0.1% streptomycin sulphate. SSCs were isolated by a two-time enzymatic digestion process as described by Izadyar et al. (2003) with some modifications. For the first enzymatic digestion, minced seminiferous tissue was suspended in DMEM containing 1 mg/ml collagenase, 1 mg/ml hyaluronidase type 2, 5 μg/ml DNase and 1 mg/ml trypsin and was incubated at 37ºC in an incubator shaker operated at 200 cycles/min for 45 min. After this, the dispersed tissue was collected and subjected to centrifugation at 1,000 rpm for 2 min. The supernatant were discarded and the tissue pellet was washed once with DMEM. For the second enzymatic digestion, the tissue was suspended in DMEM containing 1 mg/ml collagenase, 1 mg/ml hyaluronidase type 2 and 5 μg/ml DNase and was incubated in an incubator shaker operated at 200 cycles/ min for 30 min. The tissue was then centrifuged 1,000 rpm for 2 min and the supernatant was collected in a 15 ml tube. The dispersed cells, present in the supernatant were expected to contain SSCs, sertoli cells, myoid cells and other contaminating cells of the seminiferous tubular tissue. Enrichment of SSCs: For enriching the SSCs, the supernatant was filtered sucessively through a 80 μm and then 60 μm nylon net filter. The filtered cells were then transferred to BSA-lectin coated 35 mm petri dishes. The lectin-BSA coated dishes were prepared by dissolving lectin Present address: 1 Research Scholar, (fatemeh.jasour @yahoo.com), 2,4 Assistant Professor (mz1075@yahoo.com, msanjabii @gmail.com), Department of Agriculture; 3 Assistant Professor (pajhooh@yahoo.com), Department of Biotechnology; 5 Associate Professor (ofoghi@irost.ir), Department of Biotechnology. https://doi.org/10.56093/ijans.v87i8.73418