Chromosomal Integration of Transduced Recombinant Baculovirus DNA in Mammalian Cells

Abstract

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy

“…Two pairs of primers specific to two fragments on the baculoviral genome (BM3 and BM4) were designed as described. 48 PCRs were performed using 100 ng of genomic DNA and the PCR products were subjected to 0.8% agarose gel electrophoresis. 48 The FISH experiments were performed as described with minor modifications.…”

“…48 PCRs were performed using 100 ng of genomic DNA and the PCR products were subjected to 0.8% agarose gel electrophoresis. 48 The FISH experiments were performed as described with minor modifications. 20 The mitotic cells were prepared at 24 d.p.t.…”

Illegitimate DNA integration in mammalian cells

“…In some cases, however, instability appears to be restricted to the time of integration. Many studies carried out in cultured cells 41,47,48,53 and transgenic mouse lines 43 have shown that while vectors can be physically rearranged initially, expansion of cell or mouse lines reveals no further rearrangements. This leads to the conclusion that the deletions and rearrangements observed are sometimes early events in the integration process.…”

“…It appears that exogenous DNA generally integrates at one, or very few, site(s) in the recipient genome, but often in multiple copies showing the presence of 1-6 bp of microhomology or slight degrada- Illegitimate DNA integration H Würtele et al tion or addition of nucleotides at transgene-transgene junctions. [41][42][43][44][45][46][47] The NHEJ activities capable of circularizing and concatemerizing extrachromosomal DNA have been suggested to account for randomly oriented transgene arrays, while the frequent appearance of tandem arrays comprised of direct repeats is best explained by homologous recombination between circular and linear molecules before or during the integration process. 41,42 The organization and quantity of integrated DNA appear to vary according to cell type.…”

Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation

“…8 Baculoviral DNA degrades in the cells over time 13,20 and there is no evidence of baculoviral DNA integration into host chromosomes in the absence of selective pressure. 21 These attributes minimize the potential side effects and ease the safety concerns. Furthermore, the large baculovirus genome confers a huge cloning capacity of up to 38 kb 22 and baculovirus can be propagated to high titers easily by infecting its natural host insect cells.…”