Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor.
Yeast artificial chromosomes (YACs) as transgenes in transgenic animals are likely to ensure optimal expression levels. Microinjection of YACs is the exclusive technique used to produce YACs transgenic livestock so far. However, low efficiency and high cost are its critical restrictive factors. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. A targeting vector, containing the gene of interest-a human serum albumin minigene (intron 1, 2), yeast selectable marker (G418R), and mammalian cell resistance marker (neo(r)), replaced the alpha-lactalbumin gene in a 210kb human alpha-lactalbumin YAC by homogeneous recombination in yeasts. The chimeric YAC was introduced into goat fetal fibroblasts using polyethylene glycol-mediated spheroplast fusion. PCR and Southern analysis showed that intact YAC was integrated in the genome of resistant cells. Perhaps, it may offer a cell-based route by nuclear transfer to produce YACs transgenic livestock.
Zhang
Xu-Feng
XF
Shanghai Transgenic Research Center, China.
Wu
Guo-Xiang
GX
Chen
Jian-Quan
JQ
Zhang
Ai-Min
AM
Liu
Si-Guo
SG
Jiao
Bing-Hua
BH
Cheng
Guo-Xiang
GX
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism*; D019149:Bioreactors; D002459:Cell Fusion / Q000379:methods; D002478:Cells, Cultured; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D006041:Goats / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*; D007768:Lactalbumin / Q000096:biosynthesis* / Q000235:genetics; D008321:Mammary Glands, Animal / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D007768:Lactalbumin