Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method
DOI:
https://doi.org/10.17533/udea.rccp.v32n1a04Keywords:
cloning, epigenetics, lipofection, lentiviral transduction, nuclear reprogrammingAbstract
Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.
Downloads
References
Arat S, Rzucidlo SJ, Gibbons J, Miyoshi K, Stice SL. Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes. Mol Reprod Dev 2001; 60:20–26.
Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. Efficient method to generate single-copy transgenic mice by sitespecific integration in embryonic stem cells. Genesis 2006; 44:23–28.
Bertolini LR, Meade H, Lazzarotto CR, Martins LT, Tavares,KC, Bertolini, M, Murray JD. The transgenic animal platform for biopharmaceutical production. Transgenic Res 2016; 25:329-343.
Bressan FF, Dos Santos Miranda M, Perecin F, De Bem TH, Pereira FT, Russo-Carbolante EM, Alves D, Strauss B, Bajgelman M, Krieger JE, Binelli M, Meirelles FV. Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle. Cell Reprogram 2011; 13:29-36.
Bressan FF, Miranda M dos S, De Bem THC, Pereira FTVM, Binelli M, Meirelles FV. Production of transgenic animals by nuclear transfer: model for biological studies. Rev Bras Reprod Anim 2008; 32:240-250.
Cao F, Xie X, Gollan T, Zhao L, Narsinh K, Lee RJ, Wu JC. Comparison of gene-transfer efficiency in human embryonic stem cells. Mol Imaging Biol 2010; 12:15-24.
Chen T, Dent SYR. Chromatin modifiers: regulators of cellular differentiation. Nat Rev Genet 2014; 15: 93–106.
Cho J, Bhuiyan, MMU, Shin S, Park E, Jang G, Kang S, Lee B, Hwang W. Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell. J Vet Med Sci 2004; 66: 1567–1573.
Dalton AC, Barton WA. Over-expression of secreted proteins from mammalian cell lines. Protein Sci 2014; 23:517–525.
Denning W, Das S, Guo S, Xu J, Kappes JC, Hel Z. Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations. Mol Biotechnol 2013; 53:08-14.
Feng X, Cao S, Wang H, Meng C, Li J, Jiang J, Qian Y, Su L, He Q, Zhang Q. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer. Transgenic Res 2015; 24:73–85.
Fuge G, Zeng AP, Jandt U. Weak cell cycle dependency but strong distortive effects of transfection with Lipofectamine 2000 in near-physiologically synchronized cell culture. Eng Life Sci 2017; 17: 348–356.
Gong G, Dai Y, Fan B, Zhu H, Wang H, Wang L, Fang C, Wan R, Liu Y, Li R, Li N. Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle. Sci China C Life Sci 2004; 47:183–189.
Gresch O, Engel FB, Nesic D, Tran TT, England HM, Hickman ES, Hammermann R. New non-viral method for gene transfer into primary cells. Methods 2004; 33: 151-163.
Hofmann A, Kessler B, Ewerling S, Kabermann A, Brem G, Wolf E, Pfeifer A. Epigenetic regulation of lentiviral transgene vectors in a large animal model. Mol Ther 2006; 13:59-66.
Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157:1262–1278.
Jeong YH, Kim YJ, Kim EY, Kim SE, Kim J, Park MJ, Lee HG, Park SP, Kang MJ. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination. Zygote 2016; 24:442-456.
Keravala A, Calos MP. Site-specific chromosomal integration mediated by φC31 integrase. Methods Mol Biol 2008; 453:165-173.
Kues W, Niemann H. Advances in farm animal transgenesis. Prev Vet Med 2011; 102: 146-156.
Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295:868-872.
Ng RK, Gurdon JB. Epigenetic inheritance of cell differentiation status. Cell Cycle 2008 7:1173-7.
Ooi A, Wong A, Esau L, Lemtiri-Chlieh F, Gehring C. A guide to transient expression of membrane proteins in HEK-293 cells for functional characterization. Front physiol 2016; 7:300.
Rulicke T, Hubscher U. Germ line transformation of mammals by pronuclear microinjection. Exp Physiol 2000; 85:589–601.
SAS®, Statistical Analysis System. SAS/STAT User ́s Guide. Version 6.03. Cary, NC, USA: SAS Institute Inc.; 1988.
Sheng J, Wang Y, Turesky RJ, Kluetzman K, Zhang QY, Ding X. Novel transgenic mouse model for studying human serum albuminas a biomarker of carcinogenic exposure. Chemical research in toxicology 2016; 29: 797-809.
Soboleski MR, Oaks J, Halford WP. Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB J 2005; 19:440-442.
Song Y, Hai T, Wang Y, Guo R, Li W, Wang L, Zhou Q. Epigenetic reprogramming, gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor--m-carboxycinnamic acid bishydroxamide (CBHA). Protein Cell 2014; 5:382–393.
Soriano P, Gridley T, Jaenisch R. Retroviruses and insertional mutagenesis in mice: proviral integration at the Mov 34 locus leads to early embryonic death. Genes Dev 1987; 1:366–375.
Su J, Zhu L, Sherman A, Wang X, Lin S, Kamesh A., Norikane JH, Streatfield SJ, Herzog RW, Daniell H. Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. Biomaterials 2015; 70:84-93.
Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 2005; 51:187-200.
Vink T, Oudshoorn-Dickmann M, Roza M, Reitsma J-J, de Jong RN. A simple, robust and highly efficient transient expression system for producing antibodies Methods 2014; 65:5-10.
Wang Y, Shang S, Li C. Comparison of different kinds of nonviral vectors for gene delivery to human periodontal ligament stem cells. J Dental Sci 2015; 10:414-422.
Williams A, Harker N, Ktistaki E, Veiga-Fernandes H, Roderick K, Tolaini M, Norton T, Williams K, Kioussis D. Position effect variegation and imprinting of transgenes in lymphocytes. Nucleic Acids Res 2008; 36:2320-2329.
Yang P, Wang J, Gong G, Sun X, Zhang R, Du Z, Liu Y, Li R, Ding F, Tang B, Dai Y, Li N. Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin. PLoS ONE 2008; 3:e3453.
Zhang R, Yin Y, Zhang Y, Li K, Zhu H, Gong Q, Wang J, Hu X, Li N. Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle. PLoS ONE 2012; 7:e50348.
Zheng C, Baum BJ. Evaluation of promoters for use in tissue-specific gene delivery. Methods Mol Biol 2008; 434:205–219.
Published
How to Cite
Issue
Section
License
Copyright (c) 2018 Revista Colombiana de Ciencias Pecuarias
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
The authors enable RCCP to reprint the material published in it.
The journal allows the author(s) to hold the copyright without restrictions, and will allow the author(s) to retain publishing rights without restrictions.
