Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector

Abstract

This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.

Development of murine leukemia virus-based retroviral vectors with a minimum possibility of cis-activation

“…It is probably due to the fact that our final vector contains no enhancer sequence, and/or that the transcription termination signals present in the R region of both LTRs might act as a barricade, which prevent or protect the inside sequences from influencing or being influenced by the expression of the neighboring gene. 25 To prove that the newly developed vector system does not induce cancer in vivo, we are currently performing extensive experiments on mice, as recommended by the US Food and Drug Administration (http://www.fda.gov/ohrms/dockets/ac/06/ briefing/2006-4205B2_3.pdf).…”

“…These sites can be used to linearize the entire plasmid before the electroporation, which allows the whole retroviral vector DNA to integrate into the chromosome without disruption of the nucleotide sequences necessary for vector functions. 24,25 The neomycin-resistant gene cassette was also introduced, which will be used to select cells transfected with vector DNA. The final structure of plasmid containing the retroviral vector DNA was named ID (Figure 1).…”

“…Testing the possibility of cis-activation of the neighboring gene using an in vitro assay system We previously reported the development of the in vitro cell culture assay in which the effect of retroviral integration on the neighboring gene can be evaluated. 25 A newly developed vector, ROK-gp91, was tested using this assay. In this system, the retroviral vector was placed upstream from the RNA start site of the LMO2 gene whose coding sequence was replaced with the CAT sequence ( Figure 6).…”

“…The detailed procedure for this assay was previously described. 25 Briefly, the EcoRI (Klenow-blunted)-NdeI fragment of MT-gp91 was cloned to the SalI (Klenow-blunted)-NdeI site of pMT, yielding pMT-gp91. To construct the proviral form of ROK-gp91, the deleted form of 5¢LTR was amplified from plasmid ROK-gp91 using primers 5¢dLTR F (5¢-GCTAGCCCCACAACCCCTC ACTCG-3¢) and 5¢dLTR R (5¢-ACGCGTATTTTCAGACAAATACAGAAACAC AGTCAG-3¢).…”