Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

doi:10.15302/J-FASE-2016087

Front. Agr. Sci. Eng.

2016, Vol. 3

Issue (1) : 87-96 https://doi.org/10.15302/J-FASE-2016087

RESEARCH ARTICLE

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

Xiaoming HU,Jing GUO,Chunling BAI,Zhuying WEI,Li GAO,Tingmao HU,Shorgan BOU(

),Guangpeng LI(

)

Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology/Key Laboratory of Herbivore Reproductive Biotechnology and Breeding of Ministry of Agriculture, Inner Mongolia University, Hohhot 010021, China

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Abstract

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F₀, F₁ and F₂. Total muscle growth in F₀ mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

Keywords safety of transgenic bicistron gene transfer body transgenic mice muscle development

Corresponding Author(s): Shorgan BOU,Guangpeng LI

Just Accepted Date: 16 March 2016 Online First Date: 24 March 2016 Issue Date: 07 April 2016

Cite this article:

Xiaoming HU,Jing GUO,Chunling BAI, et al. Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice[J]. Front. Agr. Sci. Eng. , 2016, 3(1): 87-96.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2016087
https://academic.hep.com.cn/fase/EN/Y2016/V3/I1/87

Fig.1 Construction maps of vectors. (a) Bicistronic gene cassette; (b) pMAR-FST.

Tab.1 Primers for real-time PCR analysis

Tab.2 Comparison of transfection efficiency

Tab.3 Comparison of the integration efficiency

Fig.2 FST mRNA expression by real-time PCR

Fig.3 FST translation in bovine fetalblasts. FST protein after translation was detected by western blot assay. The mean grave value was measured by using Imagej software. A, The negative control; B, bicistronic gene cassette; C, pMAR-FST vector.

Fig.4 Transgenic cell monoclones obtained from different vectors. a, Growth state of transgenic cells under the bright-field; b, fluorescence levels of transgenic cells under the dark field. M8, M33, M36, M42, M44–45, M52–53, Monoclone of transgenic cells with pMAR-FST vector; B5–7, B11, monoclone of transgenic cells with bicistronic gene cassette.

Fig.5 PCR analysis of FST-AcGFP1 (a) and FST-MAR integration (b). Lanes assigned as: 1, M8; 2, M33; 3, M36; 4, M42; 5, M44; 6, M45; 7, M52; 8, M53; 9, control of ddH₂O; 10, negative control; 11, positive control.

Fig.6 RT-PCR analysis on RNA expressions.1, M8; 2, M33; 3, M36; 4, M42; 5, M44; 6, M45; 7, M52; 8, M53; 9, control of ddH20; 10, negative control; 11, positive control.

Fig.7 PCR analysis on FST gene in transgenic mice. M, DL2000 Marker; 1, 2, 7, 11, 17, the control of ddH₂O; 3–5, the primary generation of transgenic mice; 6, 8–10, The first generation of transgenic mice; 12–15, the second generation of transgenic mice; 16, the plasmid of positive control.

Fig.8 RT-PCR analysis on exogenous genes expressions. (a) RT-PCR analysis of FST expression; (b) RT-PCR analysis of AcGFP expression. M, 100bp Marker; 1–3, primary generation of transgenic mice; 4–7, the first generation of transgenic mice; 8–11, the second generation of transgenic mice; 12, plasmid of positive control; 13, control of ddH₂O.

Fig.9 Expressions of FST enhanced muscle development in transgenic mice. The fold changes of muscle development related gene between FST transgenic mice and wild-type mice were calculated from the results after qRT-PCR assays. (a) F₀ transgenic mice; (b) F₁ transgenic mice; (c) F₂ transgenic mice. A, MSTN; B, MyoG; C, MyoD; D, PAX3.

Fig.10 FST transgenic mice induced strong muscle development with the increased muscle bundles. (a) The body length of transgenic mice (left) and wild-type mice (right); (b) the limbs of transgenic mice (left) and wild-type mice (right); (c) histological analysis on muscle tissues from primary generation of transgenic mice; (d) histological analysis on muscle tissues from primary generation of wild-type mice.

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