Abstract

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton’s jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton’s jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p < 0.05) and LTX (19.590 ± 113,84 and 10.518 ± 442,65, respectively, p < 0.05). These results, associated with evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than other cells, independent of the kit used. Performing PCR on co-transfected cells demonstrated the presence of anti-CD3, making this approach feasible for future experiments.

摘要

不同来源的细胞在外源 DNA 的掺入和转基因细胞的形成方面表现不同。重组抗体的牛奶生产可能受益于生产转基因动物的高效转染方案。在此背景下，本研究的目的是验证来自沃顿氏胶 (MSC-WJ) 和脂肪组织 (MSC-AT) 的牛间充质干细胞的转染潜力，比较共转染方案与载体 pBC1-anti-CD3 和pEF-NEO-GFP，使用转染试剂 Lipofectamine LTX with Plus Reagent 或 Xfect。皮肤成纤维细胞（FIB）用作对照组。转染后48小时，加入新霉素并培养细胞2周。将处理过的细胞提交给荧光显微镜、流式细胞术和 PCR 评估。沃顿商学院的果冻细胞对治疗敏感并开始坏死。在流式细胞术测定中，对于 Xfect（分别为 20.057 ± 1.620,7 和 10.601 ± 702,86，p  < 0.05) 和 LTX（分别为 19.590 ± 113,84 和 10.518 ± 442,65，p  < 0.05）。这些与落射荧光评估相关的结果表明，与使用的试剂盒无关，脂肪细胞对转染的反应优于其他细胞。对共转染细胞进行 PCR 证明存在抗 CD3，使这种方法在未来的实验中可行。