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Myostatin (MSTN) was a major gene of skeletal muscle growth regulation and
a member of the transforming growth factor-β (TGF-β) super family.
It acts as a negative regulator of skeletal muscle growth. To knock out goat
MSTN gene of nuclear donor cells correctly, a gene targeting vector should be
constructed. The homologous arm of goat MSTN was amplified by PCR and restriction
enzyme digested. Intermediate vector pMD18-SA and pMD18-LA were constructed
by conventional molecular cloning method. The final target vector pLOXP-MSTN
was constructed by step-by-step cloning. The replacement type knock-out vector
was transfected into goat fetal fibroblast by electrotransfection. The homologous
arm sequences of goat MSTN including the 4.6 kb homologous long arm (LA) and
the 1.9 kb homologous Short Arm (SA) were successfully amplified using LA-PCR
technology. The LA and SA were inserted into vector pLOXP, generating MSTN gene
targeting vector pLOXP-MSTN which contained neo and tk positive-negative-selection
markers. The pLOXP-MSTN was linearized and electroporated into the nuclear donor
cells. After G418 and GANC selection, fifty-eight drug resistant cell clones
were screened and from which totally 3 positive clones were confirmed by PCR
and DNA sequencing. The constructed gene targeting vector could efficiently
target of MSTN locus and the gene knockout cell clones will be used to produce
MSTN knockout goat by means of somatic cell nuclear transplantation.
