Fast and efficient generation of recombinant baculoviruses by in vitro transposition.
A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, 151-742, Republic of Korea.
Jae Young
JY
Choi
Yang-Su
YS
Kim
Yong
Y
Wang
Xue Ying
XY
Tao
Qin
Q
Liu
Jong Yul
JY
Roh
Soo Dong
SD
Woo
Byung Rae
BR
Jin
Yeon Ho
YH
Je
D000818:Animals; D001407:Bacillus / Q000201:enzymology; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification*; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007313:Insecta; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000235:genetics
D004279:DNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012260:Ribonucleases