Elementary research into the transformation BmN cells mediated by the piggyBac transposon vector.
To generate stable transformants of Bombyx mori silkworm BmN cells continuously expressing a target gene from a piggyBac-derived vector, BmN cells were transfected with a piggyBac vector containing a neomycin-resistance gene, green fluorescent protein gene, and human insulin-like growth factor I gene (hIGF-I) and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. With the antibiotic G418, we selected stably transformed BmN cells expressing hIGF-I from the piggyBac-derived vector containing a neomycin-resistance gene driven by the ie-1 promoter from the B. mori nucleopolyhedrovirus. However, no stably transformed BmN cells transformed with the piggyBac element vector containing an SV40-promoter-driven neomycin-resistance gene were isolated. Determined with an enzyme-linked immunosorbent assay, the expression level of hIGF1 was about 7.8ng in 5x10(5) cells in which the hIGF-I gene was driven by the sericin-1 promoter, and 147.5ng in 5x10(5) cells in which the hIGF-I was under control of B. mori fibroin heavy chain gene (fib-H) promoter with its downstream signal peptide sequence. Analysis of the chromosomal insertion site by inverse PCR showed that the exogenous DNA was inserted into the cell genome randomly or at a TTAA target sequence, characteristic of piggyBac element transposition. These results are particularly important because piggyBac has been suggested for use in the transgenesis of silkworm cells.
Xue
Renyu
R
School of Medicine, Soochow University, Suzhou 215123, China.
Li
Xi
X
Zhao
Yue
Y
Xingliang
Pan
P
Zhu
Xuxian
X
Cao
Guangli
G
Gong
Chengliang
C
D000818:Animals; D001709:Biotechnology / Q000379:methods; D012831:Bombyx / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D009355:Neomycin