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. Author manuscript; available in PMC: 2020 Jul 6.
Published in final edited form as: Transgenic Res. 2017 Jun 12;26(4):477–489. doi: 10.1007/s11248-017-0025-2

Figure 2. Long Evans rat spermatogonial sub-culture and genetic modification.

Figure 2.

a) Left: Laminin-Binding spermatogenic cells illustrated in Figure 1 were used to derive Long Evans rat spermatogonial lines that develop into colonies (yellow arrows) on MEF feeder layers (cyan arrows). Scale, 40 μm. Right: Propagation of a Long Evans rat spermatogonial line during sub-culture on MEFs. Graph depicts spermatogonial yield following passages 1–5 on MEFs.

b) Flow analysis of a Long Evans rat spermatogonial line following transfection with pSBDazl-tdTomato (SB transposon) + pSBM3A (SB transposase) plasmids. Left: Red Fluorescence intensity (Region 2) in cells gated from Region 1 in adjacent forward and side scatter plots following transfection with pSBDazl-tdTomato + pSBM3A plasmids (Plus Transfection). Untransfected spermatogonia from the same culture were used to define Region 2 (Minus Transfection). Right: percent tdTomato+ cells in Region 2 scatter plots at passage 4 following co-transfection with pSBDazl-tdTomato and pSBM3A (+TF; ±SEM, n=3 transfections/spermatogonial line) -TF, Minus Transfection culture used to determine background fluorescence.