An integrase facilitates long-lasting foreign gene expression in vivo in mouse spermatogenic cells.

The objective of the present study was to attain long-lasting foreign gene expression in vivo in spermatogenic cells in the mouse testis for establishing spermatogenic-cell mediated gene transformation. Prior to in vivo gene transfer, surgical cryptorchidism was performed by retaining the testis into the abdominal cavity for 1 month to remove differentiated spermatogenic cells. Subsequently, in vivo gene transfer was conducted by electroporation with a lacZ reporter gene in combination with a retroviral integrase gene, and the testis was descended immediately to the scrotum to recover from the cryptorchidism, and restart spermatogenesis. At 1 month post-transfection in vivo, lacZ gene expression was detected in some spermatocyte-like cells in seminiferous tubules of the mouse testis. However, the recovery period of 1 month appeared to be too short, since no elongated and fully differentiated spermatids were found. At 2 months post-transfection, fully differentiated spermatogenic cells expressing the lacZ gene, albeit at low frequency, were detected when the integrase gene was co-transfected, while virtually no lacZ-positive cells were found in the absence of the integrase gene. It was concluded, therefore, that stable transformation of spermatogenic cells in vivo would be facilitated by integrase gene co-transfection.

Gene transfer using purified retroviral integrase.

We present a new method of gene transfer into cultured cells using a purified retroviral integrase protein with liposomes. The acceleration rate of transfection by the integrase was increased by a few to ten times. The integrase target sequence containing the 3' end of LTR on the introduced plasmid was necessary for the acceleration, and the orientation of this sequence determined the level of acceleration activity. The analyses of the chromosomal DNAs of each transfectant demonstrated the integration of the introduced plasmid DNA within the integrase-target sequence.

In vitro accurate transcription from the cap site of bovine leukemia virus (BLV) dependent on the BLV-infected cell nuclear lysate.

The cell-free transcriptional system initiating from the cap site in bovine leukemia virus (BLV) LTR by RNA polymerase II was constructed. The transcription was completely dependent on the template DNA and the nuclear lysate isolated from BLV-infected bat lung cells (TB1Lu). The relative transcriptional rates estimated using several deletion mutants around the promoter sequence in BLV LTR as templates closely corresponded to that obtained by transient expression assay in cultured cells using these plasmids and tax-producing plasmid. The partial purification of the factor(s) involving to the transcriptional activation from the nuclear lysate suggested that the factor(s) was different from tax and rex, the regulatory factors encoded on viral genome. The transcription from the caps site of adenovirus E3 was also stimulated in the presence of the nuclear lysate from BLV-infected cells.