Shuttle System Allowing Simplified Cloning of Expression Cassettes into Advanced Generation Lentiviral Vectors

Gruh, Ina; Schwanke, Kristin; Wunderlich, Stephanie; Blömer, Ulrike; Scherr, Michaela; Ganser, Arnold; Haverich, Axel; Martin, U

doi:10.2144/05384bm02

Cited by 4 publications

(6 citation statements)

References 10 publications

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“…TALENs are based on the NΔ134/C+17 architecture (Mussolino et al., 2011) and contain wild-type FokI nuclease domains. For generation of the eGFP donor plasmid, a 2A protease sequence (Szymczak and Vignali, 2005) was fused in frame at the beginning of an improved version of the red fluorescent protein coupled to a nuclear membrane location signal (RedStar nuc ) (Gruh et al, 2005; Okita et al., 2004). Afterward, the resulting 2A-RedStar-poly(A) expression cassette was cloned between two arms of ∼700 bp eGFP locus homology sequences in a pJet1.2 (Thermo Scientific) expression vector backbone.…”

Section: Methodsmentioning

confidence: 99%

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

et al. 2014

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SummaryGenetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

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Section: Methodsmentioning

confidence: 99%

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

et al. 2014

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“…The human CMV immediate‐early enhancer (Ce) was inserted upstream of the RRE into the backbone of pLentiShuttle, a recently developed lentiviral vector system which simplifies the insertion of transgenes 13. Several expression cassettes were introduced into pLentiShuttle with and without the CMV enhancer, respectively, to generate transfer vectors mediating the expression of GFP under control of the ubiquitous mouse PGK or cardiac human ANF‐ and human MLC2v‐, as well as lung cell‐specific human SP‐C‐ promoters.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously described ‘LentiShuttle’, a novel and efficient cloning system for lentiviral vectors 13. In this study, we used this system for the generation of vectors for stable and specific transgene expression in two different tissues.…”

Section: Discussionmentioning

confidence: 99%

“…A second‐generation, three‐plasmid packaging system was used for virus production 12. Self‐inactivating transfer plasmids were generated with the lentiviral cloning system ‘LentiShuttle’ 13, derived from pHR'SINcPPT CEW 14. The human CMV immediate‐early enhancer (positions − 598 to − 68 of the enhancer/promoter region of the major IE gene of HCMV; corresponding to positions 235–765 in pcDNA3Zeo, NCBI Accession X90639 ) was amplified by polymerase chain reaction (PCR) from the plasmid pcDNA3 using primers sense and reverse antisense introducing Eag I sites.…”

Section: Methodsmentioning

confidence: 99%

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Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Gruh

Wunderlich

Winkler

et al. 2007

The Journal of Gene Medicine

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“…For determination of transduction efficiencies, the SIVbased self-inactivating lentiviral vector plSh S EF1a hrGFP WPRE and the HIV-1-based lentiviral vector plSh H EF1a hrGFP WPRE were used. The vector plSh S is a combination of the transfer vector pSIV RMES GAE (kindly provided by Dr. Didier Nègre, É cole Normale Supérieure de Lyon, France) and the established shuttle system that has been described previously (Gruh et al, 2005). The vector plSh H belongs to the shuttle vector system described in Gruh et al (2005).…”

Section: Preparation Of Lentiviral Vector Stocks and Assessment Of Trmentioning

confidence: 99%

Induction of Pluripotent Stem Cells from a Cynomolgus Monkey Using a Polycistronic Simian Immunodeficiency Virus–Based Vector, Differentiation Toward Functional Cardiomyocytes, and Generation of Stably Expressing Reporter Lines

Wunderlich

Haase

Merkert

et al. 2012

Cellular Reprogramming

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Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

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Shuttle System Allowing Simplified Cloning of Expression Cassettes into Advanced Generation Lentiviral Vectors

Cited by 4 publications

References 10 publications

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Induction of Pluripotent Stem Cells from a Cynomolgus Monkey Using a Polycistronic Simian Immunodeficiency Virus–Based Vector, Differentiation Toward Functional Cardiomyocytes, and Generation of Stably Expressing Reporter Lines

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