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001-es BibID:	BIBFORM059252
Első szerző:	Garrels, Wiebke
Cím:	Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig Genome / Wiebke Garrels, Lajos Mátés, Stephanie Holler, Anna Dalda, Ulrike Taylor, Björn Petersen, Heiner Niemann, Zsuzsanna Izsvák, Zoltán Ivics, Wilfried A. Kues
Dátum:	2011
ISSN:	1932-6203
Megjegyzések:	Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.
Tárgyszavak:	Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban transposable elements swine animal genomics fetuses fibroblasts zygotes reporter gene assay genetically modified animals
Megjelenés:	Plos One. - 6 : 8 (2011), p. e23573-. -
További szerzők:	Mátés Lajos Holler, Stephanie Dalda Anna Taylor, Ulrike Petersen, Björn Niemann, Heiner Izsvák Zsuzsanna (1961-) (genetikus) Ivics Zoltán (1964-) (biológus) Kues, Wilfried A.
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM018216
Első szerző:	Ivics Zoltán (biológus)
Cím:	The expanding universe of transposon technologies for gene and cell engineering / Ivics Zoltán, Izsvák Zsuzsanna
Dátum:	2010
ISSN:	1759-8753
Megjegyzések:	Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Mobile DNA [electronic resource]. - 1 : 1 (2010), p. 25. -
További szerzők:	Izsvák Zsuzsanna (1961-) (genetikus)
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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001-es BibID:	BIBFORM018202
Első szerző:	Kolacsek Orsoly
Cím:	Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers / Kolacsek Orsolya, Krízsik Virág, Schamberger Anita, Erdei Zsuzsa, Apáti Ágota, Várady György, Mátés Lajos, Izsvák Zsuzsanna, Ivics Zoltán, Sarkadi Balázs, Orbán Tamás I.
Dátum:	2011
ISSN:	1759-8753
Megjegyzések:	The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo. Results We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. Conclusions We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.
Tárgyszavak:	Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:	Mobile DNA [electronic resource]. - 2 : 1 (2011), p. 5. -
További szerzők:	Krizsik Virág Schamberger Anita Erdei Zsuzsa Apáti Ágota Várady György Mátés Lajos Izsvák Zsuzsanna (1961-) (genetikus) Ivics Zoltán (1964-) (biológus) Sarkadi Balázs Orbán Tamás I.
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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