Department of Ophthalmology, Far Eastern Memorial Hospital, New Taipei City, Taiwan.
Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.
PLoS One. 2022 Mar 25;17(3):e0265183. doi: 10.1371/journal.pone.0265183. eCollection 2022.
Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics.
转基因蛋白可以通过不同的转基因系统常规地在各种哺乳动物细胞类型中表达，但转基因表达的效率受到许多因素的复杂相互作用的限制，如表达的时间一致性和与特定细胞类型（包括眼部细胞）的兼容性。在这里，我们报告了一种通过慢病毒表达系统在人角膜成纤维细胞、角膜上皮细胞和结膜上皮细胞中更有效地表达增强型绿色荧光蛋白（EGFP）的方法。首先在 HEK-293T 细胞中确定了表达 EGFP 的假病毒的相对转导单位标准。通过细胞分选可以分离出同质的 EGFP 阳性和 EGFP 阴性细胞群。根据每种细胞类型的活力曲线计算出嘌呤霉素的半最大抑制浓度（IC50）值。结果表明，在用相同数量的 EGFP 编码假病毒转导后，不同的细胞类型在 EGFP 表达效率方面存在差异。使用细胞分选仪，EGFP 阳性细胞的均匀性达到了>95%。然而，在初始分选阶段，在两轮连续培养后，经过分选的细胞中 EGFP 表达的效率明显降低，但经过多达四轮的重复分选，可获得同质的 EGFP 阳性人角膜成纤维细胞，这些细胞可以在体外连续培养中保持稳定。经分选的 EGFP 阳性细胞保持其适当的形态和细胞类型特异性蛋白表达模式。发现嘌呤霉素的耐药性取决于细胞类型，这表明必须针对每种细胞类型确定嘌呤霉素的 IC50，以确保分离出同质的 EGFP 阳性细胞。总之，重复的细胞分选是一种在连续培养中获得表达转基因蛋白的同质眼细胞群体的有效方法，而不会有抗生素潜在的混杂影响。
