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A Preliminary Study on the Knockout of Myostatinin Buffalo Somatic Cells

Author: TangGangBin
Tutor: ShiDeShun
School: Guangxi University
Course: Animal Genetic Breeding and Reproduction
Keywords: myostatin gene Knockout Liposome method Electroporation Cells transfected
CLC: S823.83
Type: Master's thesis
Year: 2005
Downloads: 137
Quote: 0
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Abstract


The buffalo livestock in southern China , with raising the level of productive forces in China , the Battle of buffalo sex being part of the machinery to replace , and buffalo meat and milk has been more and more attention . myostatin is a key genes that control muscle growth , should knock out the gene can improve the animal 's meat production performance . To improve buffalo meat with performance , cultivate new varieties of buffalo , discussed the feasibility of the research on the buffalo body cells myostatin gene knockout . First , two pairs of primers designed according to the GeneBank published cows myostatin gene sequence . Long PCR technology successfully from the local buffalo genome has been shooting with the length of homology arms length 4368bp and 1374bp . Sequencing and homology analysis showed that the length of the arm with cows corresponding sequence homology of 97 % and 96 %, indicating that the the amplified fragment source of correct completely for vector construction . The homology long arm including the myostatin gene , the first and second exons ; homologous short arm comprises a part of the third exon . Homologous to the long arm and short arm connected into pLoxp skeleton , the myostatin knock in addition to carrier - P13K constructed . Digested and PCR results prove that the carrier is constructed correctly . Then with improved tissue explant and trypsin digestion the fetus successfully isolated muscle tissue buffalo from buffalo fetal fibroblast cells cultured in vitro to the third generation . Finally, build the myostatin gene knockout vector by the liposomes law and electroporation transfection buffalo fetal fibroblast cells , the resistant cells , and transfection screening parameters and resistant cells and culture conditions were optimized to obtain of resistant cells in the PCR assays. The results showed that : ① The liposomes were transfected with liposomes 8μl, plasmid DNA2μg, and stably transfected 8h efficient . Electroporation transfection , the electric field strength 45V/mm, pulse time of 12ms , higher transfection efficiency ; ② resistant cells use DMEM 20 ? S 10μg/ml insulin 10ng/ml of bFGF 30 % conditioned medium droplet culture method cultivate better ; ③ P13K random integration occurred in the resistant cells .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Cow > Variety > Buffalo
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