The Experts below are selected from a list of 1218 Experts worldwide ranked by ideXlab platform
Shu Wang - One of the best experts on this subject based on the ideXlab platform.
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targeted transgene insertion into the aavs1 locus driven by baculoviral vector mediated zinc finger nuclease expression in human induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Shouhui Du, Rui Zhe Phang, Mohammad Shahbazi, Can Chen, Shu WangAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre Recombinase-Mediated Cassette Exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP Cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP Cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP Cassette with another transgene Cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene Exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
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efficient recombinase mediated Cassette Exchange at the aavs1 locus in human embryonic stem cells using baculoviral vectors
Nucleic Acids Research, 2011Co-Authors: Chrishan J A Ramachandra, Mohammad Shahbazi, Timothy Weixin Kwang, Yukti Choudhury, Jing Yang, Shu WangAbstract:Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated Cassette Exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate Recombinase-Mediated Cassette Exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
Aslam Abbasi Akhtar - One of the best experts on this subject based on the ideXlab platform.
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abstract pr11 madr rapid generation of somatic mosaics with locus specific stably integrated transgenic elements for generation of personalized mouse models and human organoid tumor models
Cancer Research, 2020Co-Authors: Gi Bum Kim, David Rincon Fernandez Pacheco, David Saxon, Amy Yang, Sara Sabet, Marina Dutraclarke, Rachelle Levy, Ashley Watkins, Hannah Park, Aslam Abbasi AkhtarAbstract:In situ transgenesis methods such as virus and electroporation can create somatic transgenic mice quickly, but they lack the exquisite control over copy number, zygosity, and locus specificity. We have recently established mosaic analysis by dual Recombinase-Mediated Cassette Exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. MADR provides a toolkit of elements for combinatorial labeling, inducible/reversible transgene manipulation, VCre recombinase expression, and genetic manipulation of human cells. Further, we have demonstrated the versatility of MADR by creating glioma models with mixed, reporter-identified zygosity or with “personalized” driver mutations from pediatric glioma. For example, introducing H3f3a mutation variants with MADR regulates the spatiotemporal profile of glioma, and single-cell RNA and ATAC sequencing analysis demonstrates a recapitulation of developmental hierarchy seen in K27M mutant human glioma. Moreover, we have generated novel models of supratentorial ependymoma using patient-derived oncofusion transgenes. These models display a high degree of fidelity, and we now compare these models on a single-cell level with our previous models and human tumor cell transcriptomes. In addition, we now demonstrate the ability to generalize the MADR technology to other non-CNS tissues using local plasmid delivery. Finally, we have engineered human cells to allow for MADR transgenesis and somatic transgenic organoids. These combined approaches will enable researchers to discover disease mechanisms and test therapeutics in more physiologically relevant cancer models. MADR is extensible to thousands of existing mouse lines and can be adapted to human cells, providing a flexible platform to democratize the generation of somatic transgenic disease models. This abstract is also being presented as Poster B46. Citation Format: Gi Bum Kim, David Rincon Fernandez Pacheco, David Saxon, Amy Yang, Sara Sabet, Marina Dutra-Clarke, Rachelle Levy, Ashley Watkins, Hannah Park, Aslam Abbasi Akhtar, Paul W. Linesch, Naomi Kobritz, Swasty S. Chandra, Katie Grausam, Alberto Ayala- Sarmiento, Jessica Molina, Kristyna Sedivakova, Daniel S. Gareau, Mariella G. Filbin, Serguei Bannykh, Jie Tang, Mario Suva, Bin Chen, Moise Danielpour, Joshua J. Breunig. MADR: Rapid generation of somatic mosaics with locus-specific, stably integrated transgenic elements for generation of “personalized” mouse models and human organoid tumor models [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR11.
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rapid generation of somatic mouse mosaics with locus specific stably integrated transgenic elements
Cell, 2019Co-Authors: Gi Bum Kim, David Rincon Fernandez Pacheco, David Saxon, Amy Yang, Sara Sabet, Marina Dutraclarke, Rachelle Levy, Ashley Watkins, Hannah Park, Aslam Abbasi AkhtarAbstract:In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual Recombinase-Mediated Cassette Exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Can Chen - One of the best experts on this subject based on the ideXlab platform.
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Zinc Finger Nuclease-Expressing Baculoviral Vectors Mediate Targeted Genome Integration of Reprogramming Factor Genes to Facilitate the Generation of Human Induced Pluripotent Stem Cells
Stem cells translational medicine, 2013Co-Authors: Rui Zhe Phang, Felix Chang Tay, Sal-lee Goh, Cia-hin Lau, Haibao Zhu, Wee Kiat Tan, Qingle Liang, Can ChenAbstract:Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic Cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor Cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre Recombinase-Mediated Cassette Exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.
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targeted transgene insertion into the aavs1 locus driven by baculoviral vector mediated zinc finger nuclease expression in human induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Shouhui Du, Rui Zhe Phang, Mohammad Shahbazi, Can Chen, Shu WangAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre Recombinase-Mediated Cassette Exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP Cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP Cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP Cassette with another transgene Cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene Exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
Marc F Schetelig - One of the best experts on this subject based on the ideXlab platform.
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cre lox recombinase mediated Cassette Exchange for reversible site specific genomic targeting of the disease vector aedes aegypti
Scientific Reports, 2017Co-Authors: Irina Häcker, Robert A. Harrell, Kristina L. Pilitt, Gerrit Eichner, David A Obrochta, Alfred M. Handler, Marc F ScheteligAbstract:Cre/ lox -Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti
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a functional comparison of the 3xp3 promoter by recombinase mediated Cassette Exchange in drosophila and a tephritid fly anastrepha suspensa
G3: Genes Genomes Genetics, 2013Co-Authors: Marc F Schetelig, Alfred M. HandlerAbstract:Transposable elements are widely used as vectors for integrating transgenes into the genome of insects. However, the random nature of transposon vector integrations often results in mutations and makes transgene expression subject to variable genomic position effects. This makes reliable quantitative comparisons of different transgenes difficult and development of highly fit transgenic strains laborious. Tools for site-specific transgene targeting are essential for functional genomic comparisons and to develop the most advanced transgenic insect strains for applied use. Here we describe a Recombinase-Mediated Cassette Exchange gene targeting system based on Cre/loxP that is highly efficient in Drosophila, and for the first time in a non-drosophilid, the tephritid fly, Anastrepha suspensa. This system allowed a comparison of the Drosophila constitutive polyubiquitin promoter and the artificial 3xP3 tissue-specific promoter in the same genomic context within each species, showing that the widely used 3xP3 promoter is apparently nonfunctional in the tephritid fly.
Chrishan J A Ramachandra - One of the best experts on this subject based on the ideXlab platform.
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targeted transgene insertion into the aavs1 locus driven by baculoviral vector mediated zinc finger nuclease expression in human induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Shouhui Du, Rui Zhe Phang, Mohammad Shahbazi, Can Chen, Shu WangAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre Recombinase-Mediated Cassette Exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP Cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP Cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP Cassette with another transgene Cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene Exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
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efficient recombinase mediated Cassette Exchange at the aavs1 locus in human embryonic stem cells using baculoviral vectors
Nucleic Acids Research, 2011Co-Authors: Chrishan J A Ramachandra, Mohammad Shahbazi, Timothy Weixin Kwang, Yukti Choudhury, Jing Yang, Shu WangAbstract:Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated Cassette Exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate Recombinase-Mediated Cassette Exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.