Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

Abstract

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5′- and 3′-Long Terminal Repeats (LTRs) flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3′-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3′-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed two to 20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference.

Cytometric analysis, genetic manipulation and antibiotic selection of the snail embryonic cell line Bge from Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

“…S1A) or a murine leukemia retrovirus plasmid encoding resistance to puromycin (pLNHX_cHS4_puroR) derived from pLNHX_cHS4_650 (Suttiprapa et al, 2012) by replacing the gene for neomycin resistance ( neoR ) with puroR (Supplementary Fig. S1B).…”

“…( A) Schematic of the plasmid piLenti-RNAi-GFP (Applied Biological Materials, U.S.A). (B) Schematic of the murine leukemia virus plasmid pLNHX_cHS4_650 encoding the neomycin-resistant marker (Suttiprapa et al, 2012) that was replaced with the puromycin resistant marker to engineer pLNHX_cHS4_puroR (C). MCS, multiple cloning site.…”

An antibiotic selection marker for schistosome transgenesis

“…Schistosomules were transduced with virions generated from plasmid pLNHXΔD70 (Suttiprapa et al, 2011b). Control groups of schistosomes not exposed to virions were also included in the analysis.…”

“…Accordingly, whereas the results are not directly comparable given that schistosomules unlike C. elegans do not self-replicate in culture, differences in the two models likely explain the robust enrichment of drug-resistant worms in C. elegans . Selective efficiency of the present system might be enhanced, however, by modification to the expression cassette, including incorporation of stronger promoters and/or chromosomal insulators of the transgene (Suttiprapa et al, 2011b). Furthermore, other aminoglycosides (e.g., kanamycin, gentamicin or tobramycin) may be more efficient antibiotics for selection of transgenic schistosomules.…”

“…Vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped murine leukemia virus (MLV) virions were produced in GP2-293 cells transfected with retroviral constructs, pLNHX_SmAct-Luc or pLNHXΔD70, both of which carry the neoR antibiotic resistance gene (Kines et al, 2008; Suttiprapa et al, 2011b). Viral titers were determined using two complementary approaches, a functional (biological) and second a quantitative real-time PCR (qRT-PCR; Retro-X ™ qRT-PCR Titration Kit, Clontech, USA) (Mann et al, 2011; Rinaldi et al, 2011).…”