An indicator gene to demonstrate intracellular transposition of defective retroviruses.

Abstract

An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) (6). The homology between proviruses and "retrotransposons" (1, 6) is further strengthened by the occurrence of intracellular "virus-like particles" and of intermediates of reverse transcription in the case of copia and Tyl (7-11).In mammals, analysis of transposition is difficult to perform by using the biological assays that have been developed in Drosophila or yeast (6,12). Accordingly, putative transposons have been suspected from their structural similarities with proviruses and their high copy number in the genome. In the case of the IAP sequences, transposition was demonstrated by the discovery of their integration into unusual loci (13-15), a situation reminiscent of the retrovirus insertions observed in a number of tumors (16).These observations raise a series of questions. First, how can it be demonstrated that putative transposons actually transpose in mammalian cells; in particular, are proviral copies of the retrovirus able to transpose? Second, is transposition modulated by genetic or epigenetic factors as observed in Drosophila and yeast (6,(17)(18)(19)? Third, can transposition be implicated in tumorigenic processes?In addressing these questions, the potentially low frequency of transposition has to be taken into account (see refs. 20 and 21 for yeast and Drosophila). To analyze transposition in mammalian cells, we therefore constructed an "indicator gene for retrotransposition," which should allow the detection of transposition (by selective procedures) for any genetic element that transposes via an RNA intermediate. MATERIALS AND METHODSPlasmid Construction. To construct the retrotransposition indicator (neo)RT, the sequence for polyadenylylation of the thymidine kinase (tk) gene of the herpes simplex virus was isolated from plasmid pAGO (22) as a Sma I/Nco I 310-basepair (bp) fragment and was inserted at the unique BamHI site of pZip plasmid (23) between the Moloney murine leukemia virus (Mo-MLV) donor and acceptor splice sites after Klenow enzyme treatment of both fragments. The fragment containing the polyadenylylation sequence and the splice sites was next isolated as a Kpn I/Kpn I fragment and inserted between the SVtk promoter and the coding region of the neo gene at the Bgl II site of pSVtkneop3 (24) after Klenow treatment of both fragments; the neoRT indicator gene (Fig. L4) was then isolated as a HindIII/Sma I fragment.To delete the env genes in pMov3 (25) The final construction [pMo-MLV(neo)RTI was obtained upon ligation of the deleted pMov3 vector with the (neo)RT indicator gene after Klenow treatment (Fig. 1B).Cell Culture, Transfection, and Infection. Methods are described in ref. 27. Cells used were NIH 3T3 and FG10 (28), and the viral producer lines 3T3 Mo-MLV for Moloney and E404B for . 3T3 LacZ cells were a gift from D. Rocancourt and C. Bonnerot (Institut Pasteur). Selection of G418-resistant cells ...

LINE-1 Retrotransposable Element DNA Accumulates in HIV-1-Infected Cells

“…2A) (6,7,63). Previously, this assay has been carried out in either 293T or HeLa cells, both of which can be transfected at high efficiency by the Ͼ18-kb L1 retrotransposition assay plasmid.…”

“…The in vitro L1 retrotransposition assay is a modified version of a method which has been described elsewhere (6,7,63). Jurkat cells were maintained in RPMI 1640-15% FBS.…”

Improvement of HIV-1 and Human T Cell Lymphotropic Virus Type 1 Replication-Dependent Vectors via Optimization of Reporter Gene Reconstitution and Modification with Intronic Short Hairpin RNA

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LINE-Like Retrotransposition inSaccharomyces cerevisiae

“…Only after scZorro3 transcription, splicing, and reverse trascription/integration does the marker produce functional HIS3 protein. This assay is based on previously described assays in yeast, mouse, and human (Heidmann et al 1988;Curcio and Garfinkel 1991;Moran et al 1996). (C) mHIS3AI-tagged Ty1, empty vector, or scZorro3 on 2m vectors were transformed into strain GRF167 and individual clones were induced on SC ÀURA 1galactose plates for 3 days at 23°, then replica plated to SC ÀHIS plates.…”