Hyperactive self-inactivating
            <i>piggyBac</i>
            for transposase-enhanced pronuclear microinjection transgenesis

Marh, Joel; Stoytcheva, Zoia; Urschitz, Johann; Sugawara, Atsushi; Yamashiro, Hideaki; Owens, Jesse B.; Stoytchev, Ilko; Pelczar, Pawel; Yanagimachi, Ryuzo; Moisyadi, Stefan

doi:10.1073/pnas.1216473109

Cited by 46 publications

(62 citation statements)

References 43 publications

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“…This conclusion is supported by recent evidence that truncated polyQ domains containing three glutamine blocks are sufficient to enable Sry to induce Sox9 expression in a rat pre-Sertoli cell line (9). (22,23) in which the expression of the transgene is controlled by a constitutively active human ubiquitin C (UBC) promoter (Fig. 5A).…”

Section: A Truncated Domesticus-type Polyq Domain Stabilizes Sry Proteinsupporting

confidence: 68%

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

Zhao

Davidson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.sex development | Y chromosome | testis | TESCO

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Section: A Truncated Domesticus-type Polyq Domain Stabilizes Sry Proteinsupporting

confidence: 68%

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

Zhao

Davidson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…2). Both PNI and the CPI methods were successfully employed with SB, PB and Tol2 transposon components for germline transgenesis in fish [103], frogs [92], mice [10,104], rats [105] and domestic pigs [65,87]. A significant increase in the ratio of transgenic animals per microinjected zygotes has been consistently reported.…”

Section: Application Of Transposon Systems For Genetic Engineeringmentioning

confidence: 96%

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

Bosch

Forcato

Alustiza

et al. 2015

Cell. Mol. Life Sci.

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Transgenic farm animals are attractive alternative mammalian models to rodents for the study of developmental, genetic, reproductive and disease-related biological questions, as well for the production of recombinant proteins, or the assessment of xenotransplants for human patients. Until recently, the ability to generate transgenic farm animals relied on methods of passive transgenesis. In recent years, significant improvements have been made to introduce and apply active techniques of transgenesis and genetic engineering in these species. These new approaches dramatically enhance the ease and speed with which livestock species can be genetically modified, and allow to performing precise genetic modifications. This paper provides a synopsis of enzyme-mediated genetic engineering in livestock species covering the early attempts employing naturally occurring DNA-modifying proteins to recent approaches working with tailored enzymatic systems.

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“…These same investigators additionally compared a hyperactive version of the pBt with the most active SB transposase version (SB100X) with similar conclusions [19,21]. Both SB100X and the hyperactive pBt have now been applied to mammalian transgenesis, demonstrating germline transmission of the transgene [17,22]. In the case of SB, donor plasmid is coinjected into zygote pronuclei together with mRNA coding for the SB100X transposase [22].…”

Section: Introductionmentioning

confidence: 96%

“…For pBtmediated transgenesis, we developed an alternative approach, where the helper and donor plasmid components were incorporated into a single plasmid construct known as pmGENIE-3 [23]. Recently, these pBt-based self-inactivating plasmids with a large cargo capacity were additionally engineered to contain the hyperactive form of pBt and with an antibiotic-free selection in bacteria (pmhyGENIE-3) [17] to conform to regulatory recommendations for the elimination of antibiotic resistance markers during transgenesis [24]. In comparison to SB100X, due to the high transposition efficiency of the pBt, it is not necessary to use its mRNA for effective transgenesis.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

Zeng

Meng

et al. 2014

Biology of Reproduction

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The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

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Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

Cited by 46 publications

References 43 publications

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

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