Effects Of Lentivirus-Mediated Shrna Silencing Of Tgf¦Â1 In Mouse Hepatic Stellate Cells On The Expression Of ¦¡1 Type I Collagen
Keywords
Transforming Growth Factor Beta, Type I Collagen Alpha, Rna Interference, Small Molecules, Lentiviral Vectors
Abstract
Objective To construct a rat transforming growth factor ¦Â1 (TGF¦Â1) gene RNA interference lentiviral vector, and observe its TGF¦Â1 gene silencing effect on rat hepatic stellate cells HSC-T6 and its effect on type ¢ñ collagen ¦Á1 (alpha-1type ¢ñcollagen, Col1¦Á1 ) expression. Methods Based on the rat TGF¦Â1 gene CDs sequence, three interference targets were screened, three pairs of small interfering RNA (siRNA) were synthesized, each pair of siRNA was transferred into HSC-T6 cells, and RT-PCR was used to screen the three pairs of siRNA. Find the best siRNA, design and synthesize shRNA targeting the best siRNA target sequence, anneal to form double strands, connect to the vector pGreenPuro, construct the pGreenPuro/TGF¦Â1 shRNA lentiviral vector, and perform enzyme digestion and sequencing identification. The pGreenPuro/TGF¦Â1 shRNA lentiviral vector is packaged by 293 cells, and the highly infectious lentiviral particles produced by the packaging are infected into HSC-T6 cells. The GFP expression of the infected HSC-T6 cells is observed under an inverted microscope, and the recombination is detected at the mRNA and protein levels. The silencing effect of lentiviral pGreenPuro/TGF¦Â1shRNA on the TGF¦Â1 gene of HSC-T6 cells and its effect on the expression of Col1¦Á1. As a result, the best interference target sequence of TGF¦Â1 gene was screened. The results of enzyme digestion and sequencing confirmed that the pGreenPuro/TGF¦Â1 shRNA lentiviral vector was successfully constructed. After HSC-T6 cells were infected with pGreenPuro/TGF¦Â1 shRNA lentivirus for 48 hours, RT-PCR detection showed no expression of TGF¦Â1 gene and weak expression of Col1¦Á1 gene; Westernblot detection showed that the expression of TGF¦Â1 and Col1¦Á1 proteins was very weak. Conclusion A rat TGF¦Â1 gene RNA interference lentiviral vector was successfully constructed, which can effectively silence the TGF¦Â1 gene of HSC-T6 cells and inhibit the expression of Col1¦Á1.
For further information of this article and research, feel free to contact our team for asssitance.
Original research was done by Zhang Ronghua, Yan Guohe, Wang Guojian, Su Yongping
About ETChem
ETChem, a reputable Chinese Collagen factory manufacturer and supplier, is renowned for producing, stocking, exporting, and delivering the highest quality collagens. They include marine collagen, fish collagen, bovine collagen, chicken collagen, type I collagen, type II collagen and type III collagen etc. Their offerings, characterized by a neutral taste, and instant solubility attributes, cater to a diverse range of industries. They serve nutraceutical, pharmaceutical, cosmeceutical, veterinary, as well as food and beverage finished product distributors, traders, and manufacturers across Europe, USA, Canada, Australia, Thailand, Japan, Korea, Brazil, and Chile, among others.
ETChem specialization includes exporting and delivering tailor-made collagen powder and finished collagen nutritional supplements. Their extensive product range covers sectors like Food and Beverage, Sports Nutrition, Weight Management, Dietary Supplements, Health and Wellness Products, ensuring comprehensive solutions to meet all your protein needs.
As a trusted company by leading global food and beverage brands and Fortune 500 companies, ETChem reinforces China’s reputation in the global arena. For more information or to sample their products, please contact them and email karen(at)et-chem.com today.
