J Vet Sci.  2016 Jun;17(2):145-152. 10.4142/jvs.2016.17.2.145.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

• He XY
• Ma LB
• He XN
• Si WT
• Zheng YM

• KMID: 2413163
• DOI: http://doi.org/10.4142/jvs.2016.17.2.145

Abstract

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

Keyword

bovine; human telomerase reverse transcriptase; mammary gland epithelial cells; somatic cell nuclear transfer

MeSH Terms

Adenoviridae/genetics
Animals
Animals, Genetically Modified/genetics/metabolism
Cattle
Cloning, Organism/veterinary
Embryonic Development
Female
*Gene Expression
Green Fluorescent Proteins/genetics/metabolism
Mammary Glands, Animal/cytology/*metabolism
Muramidase/*genetics/metabolism
Nuclear Transfer Techniques/*veterinary
Recombinant Proteins/genetics/metabolism
Telomerase/*biosynthesis/genetics
Transfection/veterinary
Recombinant Proteins
Green Fluorescent Proteins
Telomerase
Muramidase

Figure

• 

  Fig. 1 Schematic representation of a plasmid pEBH. Diagrammatic representation of the co-expression construction of pEBH contained the 5' flanking fragment (BBC5) and 3' flanking fragment (BBC3) of bovine β-lactoglobulin, human lysozyme (hLYZ), the CMV promoter, green fluorescent protein (GFP), and the neomycin/kanamycin resistance gene (Neo).

• 

  Fig. 2 Detection of transgenic efficiency of hTERT-bMGEs and bMGEs by fluorescence images and FACS analysis. hTERT-bMGEs group was a mammary gland specific expression vector (pEBH) encoding human lysozyme (hLYZ) and GFP transfer into hTERT-bMGEs, which was obtained by transfecting the adenovirus-mediated hTERT gene into bovine mammary gland epithelia cells (bMGEs), with the bMGEs used as a control. (A–C) Phase contrast, fluorescence images and FACS of bMGEs at 72 h post-transfection with plasmid pEBH. (D–F) The transfection efficiency of the hTERT-bMGEs group was much higher than that of the bMGEs group based on fluorescence detection. 100× (A, B, D and E).

• 

  Fig. 3 (A) RT-PCR for the detection of hLYZ mRNA in hLYZ-positive hTERT-bMGEs. Lane M, 200 bp Ladder; Lane 1, the positive in hTERT-bMGEs (850 bp); Lane 2, the negative in non-transgenic hTERT-bMECs. (B) Western blot analysis of antibody specificity in hLYZ-positive hTERT-bMGEs. Expression of hLYZ in positive hTERT-bMGEs was shown by Western blot (+), but no expression of non-transgenic hTERT-bMECs was observed (-). Expression of β-actin in hLYZ-positive hTERT-bMGEs and non-transgenic hTERT-bMECs was shown by Western blot analysis.

• 

  Fig. 4 The development of NT-Embryoes from hTERT-bMGEs used as donor cells. (A–G) Substantially synchronous cell divisions were shown from the 2-, 4-, 8-, 16-cell stage, stagmorula and blastocyst to the hatched blastocysts stage. (H–J) Phase contrast of blastocysts derived from hTERT-bMGEs-18, hTERT-bMGEs-28 and bMGEs-10 used as donor cells. (K and M) Phase contrast and fluorescence images of blastocyst derived from hLYZ-positive hTERT-bMGEs used as donor cells. (L and N) Phase contrast and fluorescence images of hatched blastocysts derived from hLYZ-positive hTERT-bMGEs used as donor cells. (A–N) 100×.

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