Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

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.

Keywords:

• Transfection
• pBC1
• humanized anti-CD3
• monoclonal antibody

Additional information

Funding

The authors acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa do Distrito Federal [FAP-DF; N° 0193.001479/2016], Financiadora de Estudos e Projetos (FINEP) for financial support (Embrapa SEG 0.11.13.06.001.06.03).