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.
Highlights
Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs)
simian virus 40 large T antigen (SV40LT) Leads to Extension of Canine Fibroblast Cell Lifespan without Inducing Cancerous Properties Our primary fetal canine fibroblast line, K9 fetus 1, had a very short cellular lifespan, showing the senescence phenotype at approximately passages 5–7 (Fig. 1a). The growth of these cells was nearly halted after passage 13, Fig. 1 Immortalization of canine primary fibroblast cells via ectopic expression of SV40LT. a Cell growth rates of different passages of K9 fetus 1 fibroblast cells were examined by counting 3 days after plating (1 × 105). b Western blotting analysis showing expression of SV40LT in control K9 fetus 1 fibroblast cells and cells expressing SV40LT. β-Actin was used as a loading control. c Cumulative growth curves of control K9 fetus 1 fibroblast cells and cells expressing SV40LT. d Microscopic images showing cellular morphology of control K9 fetus 1 fibroblast cells and cells expressing SV40LT
Because a previous study demonstrated that SV40LT enabled conversion of some types of normal cells into cancerous cells [20], we examined whether SV40LT-overexpressing K9 fetus 1 cells showed cancer cell properties by comparison with SV40LT-overexpressing K9 fetus 1 cells transduced with H-RASV12, an oncogenic mutant of H-RAS (Fig. 2a)
Summary
Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). Somatic cell nuclear transfer (SCNT) is an animal cloning technique that involves implanting a somatic cell into an enucleated oocyte [1] This method has been remarkably improved and applied in various mammals, including cattle, goat, pig, horse, cat, and dog, since the. Primary animal somatic cells, which are used as nucleus donors, show a finite cellular lifespan in vitro; at the end of this lifespan, the cell enters a state of irreversible replicative senescence [10]. This phenomenon was first reported by Hayflick and found to be related to the “end replication problem,” which leads to telomere shortening with each cell division [11, 12]. SV40LT does not disturb cellular reprogramming or dedifferentiation from specific somatic cells up to the pluripotent embryo stage
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