Optimization of in vitro culture and transfection condition of bovine primary spermatogonial stem cells

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Abstract

The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro- and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 µg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 µg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 µg/mL of vitamin C or 25 µg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox.

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Genetic transformation of mouse embryos by microinjection of purified DNA.

Matthew W. Gordon, Gordon D. Plotkin, F Ruddle … (1980)

A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.

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Efficient transfection method for primary cells.

Nicole Krott, Rüdiger Blindt, I Breibach … (2002)

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.

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Jürgen Wolf, Gregor Siebenkotten, Günter Kraus … (2004)

The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons. Copyright 2003 Elsevier Inc.

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Author and article information

Contributors

A. Jafarnejad: Role: ND

M. Aminafshar: Role: ND

M. Zandi: Role: ND

M. R. Sanjabi: Role: ND

N. Emamjomeh Kashan: Role: ND

Journal

Journal ID (publisher-id): sajas

Title: South African Journal of Animal Science

Abbreviated Title: S. Afr. j. anim. sci.

Publisher: The South African Society for Animal Science (SASAS) (Pretoria, Gauteng, South Africa )

ISSN (Print): 0375-1589

ISSN (Electronic): 2221-4062

Publication date (Print and electronic): 2018

Volume: 48

Issue: 1

Pages: 108-116

Affiliations

[01] Tehran orgnameIslamic Azad University orgdiv1Faculty of Agriculture and Natural Resources, Science and Research Branch orgdiv2Department of Animal Science Iran

[02] Tehran orgnameIranian Research Organization for Science and Technology orgdiv1Department of Agriculture Iran