Counterselection and Co-Delivery of Transposon and Transposase Functions for Sleeping Beauty-Mediated Transposition in Cultured Mammalian Cells

Harvard University, Cambridge, Massachusetts, United States
Bioscience Reports (Impact Factor: 2.64). 12/2005; 24(6):577-94. DOI: 10.1007/s10540-005-2793-9
Source: PubMed


Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

Download full-text


Available from: Lalitha R Belur
  • Source
    • "In contrast to random recombination, insertion mediated by transposition occurs without altering the flanking chromosomal sequence. The SB transposon system has been used for stable genetic modification of multiple rodent and human cell lines [16] [17] [19] and primary cells including mouse liver [20] [21] [22] [23] [24] [25], human skin cells [26], mouse lung [27] [28] [29], and human peripheral blood T-cells [30] as well as embryonic stem (ES) cells derived from mice [31] [32] and humans [33] [34]. Murine MAPC modified using an SB transposon engineered for expression of a dual reporter encoding DsRed2 and firefly luciferase have been used to study the homing pattern of MAPC via in vivo bioluminescence imaging after transplant into immunodeficient mice [35]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Non-viral integrating systems, PhiC31 phage integrase (ϕC31), and Sleeping Beauty transposase (SB), provide an effective method for ex vivo gene delivery into cells. Here, we used a plasmid-encoding GFP and neomycin phosphotransferase along with recognition sequences for both ϕC31 and SB integrating systems to demonstrate that both systems effectively mediated integration in cultured human fibroblasts and in rat multipotent adult progenitor cells (rMAPC). Southern blot analysis of G418-resistant rMAPC clones showed a 2-fold higher number of SB-mediated insertions per clone compared to ϕC31. Sequence identification of chromosomal junction sites indicated a random profile for SB-mediated integrants and a more restricted profile for ϕC31 integrants. Transgenic rMAPC generated with both systems maintained their ability to differentiate into liver and endothelium albeit with marked attenuation of GFP expression. We conclude that both SB and ϕC31 are effective non-viral integrating systems for genetic engineering of MAPC in basic studies of stem cell biology.
    Full-text · Article · Oct 2011
  • Source
    • "Fifth, the frequency of random integration is cell type specific. For example, McIvor and colleagues reported that NIH3T3 cells had a six times higher stable baseline transfection frequency relative to HeLa cells (0.15 vs. 0.025%, respectively) as defined by transfection with pT2/neo (SB transposon encoding the neomycin resistance gene) alone (Converse et al., 2005). In similar studies using an unrelated nonviral integrating vector system, the Calos group also demonstrated an *31-fold higher integration frequency in NIH3T3 cells than in 293 cells (0.25 vs. 0.008%) as defined by transfection with bacteriophage FC31 cargo-containing vector without integrase (Thyagarajan et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We have shown that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery (i.e., transposon and transposase are on separate plasmids) is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilization, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed(+) T cell clones were generated and found to be negative for the transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was positive for transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007-0.047 per cell) by quantitative PCR. Transposase-positive bulk T cells lacked transposase plasmid demonstrated by Hirt (episomal) extracted DNA and showed no detectable transposase by Southern hybridization, Western blot, and quantitative RT-PCR analyses. Cytogenetic and array comparative genomic hybridization analyses of the only identified transposase-positive clone (O56; 0.867 copies per cell) showed no chromosomal abnormality or tumor formation in nude mice although transposon remobilization was detected. Our data suggest that SB delivery via plasmid in T cells should be carried out with caution because of unexpectedly high copy numbers of randomly integrated SB transposase.
    Full-text · Article · Nov 2010 · Human gene therapy
  • Source

    Preview · Article ·
Show more