SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

Abstract

BackgroundSomatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.ResultsSV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.ConclusionsAlthough lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

The proliferative lifespan of adult sheep fibroblasts can be conditionally extended by tetracycline-inducible expression of human telomerase reverse transcriptase

“…Previous studies suggested that long-term cultured primary donor cells yielded lower blastocyst formation, probably due to the senescence of cells (Lan et al 2006;Liu et al 2016). Although the lifespan of cells could be extended by simian virus 40 large T antigen (SV40LT), the SCNT embryos derived from those cells exhibited a very restricted development (Eun et al 2017). In the present study, the enhanced blastocyst development of A3h38(?…”

Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer

“…To overcome this obstacle in the generation of transgenic model, and to determine if commonly used promoters could be efficient in canine models, we compared the hEF1α and CMV promoter sequence by detecting EGFP transgene in SCNT-mediated transgenic dogs. Due to the short life span of canine fibroblasts, conducting SCNT using a heterogeneous donor cell population right after the transfection was the only option to produce transgenic dogs [29,30]. Our results clearly showed that the CMV promoter…”

Generation of reproductive transgenic pigs of a CRISPR‐Cas9‐based oncogene‐inducible system by somatic cell nuclear transfer

“…Previously, porcine cells were effectively transformed into cancerous cells by an ectopic expression of both simian virus 40 large T (SV40LT) antigen, which was used to inactivate p53‐ and RB1‐mediated tumor‐suppressive pathways, and oncogenic HRAS G12V . [ 19 ] Here, we established an inducible expression system of these oncogenes using CRISPR‐Cas9. To establish an efficient multigene expression system, we constructed a 2A peptide‐mediated polycistronic gene expression cassette, DsRed‐P2A‐ SV40LT‐T2A‐HRAS G12V (DSH) (Figure S1A).…”

“…The primary pig fibroblasts used in this study were obtained from the skin tissue of a male Yucatan miniature pig as previously described. [ 19 ] The cells were maintained in complete medium: Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% v/v fetal bovine serum, 1% v/v Gluta‐max, 1% v/v Nonessential amino acids, 1% v/v Antibiotic–Antimycotics, and 0.1% v/v 2‐mercaptoethanol, all obtained from Life Technologies, CA, USA. The incubation conditions for the primary culture were 37°C and 5% CO 2 in a humid incubator.…”