Recombinant adenovirus‐mediated human telomerase reverse transcriptase gene can stimulate cell proliferation and maintain primitive characteristics in bovine mammary gland epithelial cells

Abstract

The human telomerase reverse transcriptase (hTERT) gene has been used to stimulate the proliferation of most types of human cells. The present study was designed to evaluate the feasibility and efficiency of adenovirusmediated hTERT in the proliferation of bovine mammary gland epithelial cells (bMGEs). A plasmid and an adenovirus vector that carried hTERT, namely pEGFP-hTERT and Ad-hTERT, were constructed and transfected into bMGEs, respectively. In order to select the best strategy for stimulating cell proliferation, the adenovirusand plasmid-mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The results showed that only Ad-hTERT had high infection efficiency and produced a positive polyclone population (hTERT-bMGEs). The characteristics of the hTERT-bMGEs were investigated with further analysis by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, proliferation assays, and flow cytometry, which showed that hTERT facilitated strong cell proliferation. Real-time quantitative PCR showed a normal level of expression of beta-casein, the caspase-8 and c-myc proto-oncogene, and immunofluorescence demonstrated the properties of the epithelial cells. In conclusion, the adenovirus-mediated hTERT gene could not only extend the cell lifespan, but also maintained the primary characteristics of the cells. It may be possible to extend the use of a wide variety of non-human mammalian cells in this way. This study has provided additional insight into the mechanism of cell proliferation by demonstrating the lack of integration of the adenovirus-mediated hTERT gene into the mammalian genome.

Intervention of rAAV-hTERT-Transducted Nucleus Pulposus Cells in Early Stage of Intervertebral Disc Degeneration: A Study in Canine Model

“…The c-myc and caspase-8 genes also remain constant in the transducted cells at early and late passages, which indicated that no major genetic mutations occurred in the transducted cells. 34 The limitations of this study included: (1) the transducted cells were not labeled before injection process; (2) the duration of this study was too short to observe the efficacy and cell proliferation capacity of rAAV-hTERTtransducted cells in the long run; (3) the tumor genes and tumor repression genes, related with the hTERT, were not studied.…”

Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf

“…Ovine endometrial cell lines immortalized with HPV E6/E7 genes were also shown to retain original cell phenotype (Johnson et al 1999 ). Human cytotrophoblast cells and bovine mammary gland epithelial cells (bMGEs) immortalized with hTERT gene not only showed enhanced lifespan but also retained original parental phenotype (He et al 2011 ; Wang et al 2006 ).…”

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cellsin vitromodel

“…The positive- hLYZ hTERT-bMGEs were obtained under G418 selection, after which the total cell lysates were subjected to SDS-PAGE in 12% polyacrylamide gel. Proteins were transferred onto PVDF membrane (Millipore, USA) and probed with anti- hLYZ (Sigma) and anti-β-actin (AC-15; Sigma) diluted 1 : 100 and 1 : 500, after which secondary antibody (Sigma) was performed at a 1 : 1,000 dilution [ 10 ]. Immunoreactive proteins were detected using an ECL detection system (Amersham, USA).…”

“…Therefore, it is necessary to seek a more efficient strategy for mammary cells as nuclei donors to solve this problem. We previously reported an in vitro bovine mammary gland cells model (hTERT-bMGEs) in which an adenovirus vector carrying human telomerase reverse transcriptase (hTERT) was constructed and infected into bovine mammary gland epithelia cells (bMGEs) [ 10 ]. Our results confirmed that the adenovirus disappeared from the hTERT-bMGEs at passage 15 via successive detection of green fluorescent protein (GFP) and Western blot because of the characteristics of the unintegrated adenovirus.…”

“…However, the lifespan of the hTERT-bMGEs was prolonged to more than passage 40 as a result of the initial effect of hTERT gene. The hTERT-bMGEs not only extended the cell lifespan, but also maintained the primary characteristics of the bMGEs [ 10 ]. Therefore, it provided a good cell model for subsequent research.…”