|
|
Comparison of the Expression Levels of EGFP Gene Driven by CMV Promoter in Different Tissues of the Transgenic Pigs (Sus scrofa) and Mice (Mus musculus) |
|
|
Abstract The CMV promoter is often used as a transcriptional regulatory sequence in eukaryotic expression vectors, and considered as a universal element capable of transcriptional activity in a wide rang of cells in vitro. However, reports documenting the transcriptional activity of this control element in different tissues of transgenic animals are relatively less. In this study, we produced the CMV-EGFP integrated transgenic pigs (Sus scrofa) through somatic cell nuclear transfer technique and CMV-EGFP integrated transgenic mice (Mus musculus) through pronuclear microinjection. Transgenic positive animals were identified through PCR screen. Then the expression level of reporter gene EGFP was investigated in liver, heart and muscle of the transgenic pigs through qPCR assay. The results showed that EGFP is most abundant in heart, and modest in muscle, and relatively less abundant in liver. We further examined the expression pattern of EGFP in a variety of tissues in both transgenic pigs and mice through detecting the fluorescence signal from cryosections. The results showed that the CMV promoter can drive EGFP expression in all the examined tissues of transgenic pigs and mice, but on closer inspection, we found that the fluorescence intensity of EGFP was obviously different in different tissues, where the signal was most strong in lung, small intestine and pancreas, modest in muscle and heart, and weak in spleen, kidney and liver of transgenic pigs. Similarly, expression level of EGFP was highest in lung, modest in brain, liver and muscle, and lower in spleen and kidney of transgenic mice. These experiment results suggested that the CMV promoter could drive the expression of EGFP in a wide range of tissues, however, its activity varies notably across different tissue, where it is most active in lung, modest active in muscle, and less active in kidney and spleen in both the transgenic pigs and transgenic mice. This study indicates that the abundance of transcription factors capable of activating the CMV promoter varies significantly in different tissues of animals. Therefore, whether to use the CMV promoter for establishing a transgenic animal model, should be base on the targeted expression site of the extragenous gene in the animal.
|
Received: 03 March 2015
Published: 24 July 2015
|
|
|
|
[1]Brooks A R, Harkins R N, Wang P, et al. Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle [J].The Journal of Gene Medicine, 2004, 6(4):395-404[2]Chalfie M, Tu Y, Euskirchen G, et al.Green fluorescent protein as a marker for gene expression[J].Science, 1994, 263(5148):802-805[3]Collas P.Modulation of plasmid DNA methylation and expression in zebrafish embryos[J].Nucleic Acids Research, 1998, 26(19):4454-4461[4]Foecking M K, and Hofstetter H.Powerful and versatile enhancer-promoter unit for mammalian expression vectors[J].Gene, 1986, 45(1):101-105[5]Furch P A, L Hennighausen, C Baker, et al.The variability in activity of the universally expressed human cytomegalovirus immediate early gene 1 enhancerpromoter in transgenic mice[J].Nucleic Acids Research, 1991, 19(22):6205-6208[6]Gruh I, Wunderlich S, Winkler M, et al.Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors[J].The Journal of Gene Medicine, 2008, 10(1):21-32[7]Kato M, Yamanouchi K, Ikawa M, et al.Efficient selection of transgenic mouse embryos using EGFP as a marker gene[J].Molecular Reproduction and Development, 1999, 54(1):43-48[8]Kong Q, Wu M, Huan Y, et al.Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs[J].PLoS One, 2009, 4(8):e6679-e6679[9]Massie B, Mosser D D, Koutroumanis M, et al.New adenovirus vectors for protein production and gene transfer[J].Cytotechnology, 1998, 28(1-3):53-64[10]McGowan R, Campbell R, Peterson A, et al.Cellular mosaicism in the methylation and expression of hemizygous loci in the mouse[J].Genes & Development, 1989, 3(11):1669-1676[11]McGrew M J, Sherman A, Ellard F M, et al.Efficient production of germline transgenic chickens using lentiviral vectors[J].EMBO Reports, 2004, 5(7):728-733[12]Prosch S, Stein J, Staak K, et al.Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro[J].Biological Chemistry Hoppe-Seyler, 1996, 377(3):195-201[13]Schmidt E V, Christoph G, Zeller R, et al.the cytomegalovirus enhancer: apan-active control enlment in transgenic mice[J].Molecular and Cellular Biology:., 1990, 10(0):4406-4411[14]Tessitore A, Faella A, O'Malley T, et al.Biochemical, pathological, and skeletal improvement of mucopolysaccharidosis VI after gene transfer to liver but not to muscle[J].Molecular Therapy, 2008, 16(1):30-37[15]Tsien R Y.The green fluorescent protein[J]. , .[J].Annual Review of Biochemistry, 1998, 67(0):509-544[16]Vasey D B, Lillico S G, Sang H M, et al.CMV enhancer-promoter is preferentially active in exocrine cells in vivo[J].Transgenic Research, 2009, 18(2):309-314[17]Whitelaw C B, Radcliffe P A, Ritchie W A, et al.Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector[J].FEBS Letters, 2004, 571(1-3):233-236 |
|
|
|