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1.  Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression 
Nucleic Acids Research  2004;32(12):e106.
Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmySΔS, EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-α) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (PPIR8) placed 5′ of desired transgenes; (iii) within modified enhancer-free 3′-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct PPIR-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines.
doi:10.1093/nar/gnh104
PMCID: PMC484201  PMID: 15258250
2.  Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction 
Nucleic Acids Research  2002;30(21):e113.
In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult-to-transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit-like self-inactivating lentiviral expression vector family which is compatible to state-of-the-art packaging and pseudotyping technologies and contains, besides essential cis-acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate-early promoter (PhCMV) or the human elongation factor 1α (PhEF1α), (iii) PhCMV– or PPGK– (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein-based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size-optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R-U5-ψ+-SD, cPPT-RRE-SA and 3′LTRΔU3 modules or placed within the 5′U3 (–78 bp) and 3′LTRΔU3 (8666 bp). pLentiModule enables straightforward cassette-type module swapping between lentiviral expression vector family members and facilitates the design of Tat-independent (replacement of 5′LTR by heterologous promoter elements), regulated and self-excisable proviruses (insertion of responsive operators or LoxP in the 3′LTRΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi-purpose construction kit-like vector series described here is compatible with itself as well as many other (non-viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human diseases.
PMCID: PMC135834  PMID: 12409472
3.  Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells 
Nucleic Acids Research  2001;29(4):e19.
We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.
PMCID: PMC29626  PMID: 11160939

Results 1-3 (3)