Tn5 Transposase-Mediated Mouse Transgenesis1

Suganuma, Ryota; Pelczar, Pawel; Spetz, Jean François; Höhn, Barbara; Yanagimachi, Ryuzo; Moisyadi, Stefan

doi:10.1095/biolreprod.105.044669

Cited by 39 publications

(35 citation statements)

References 40 publications

(79 reference statements)

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“…The procedures were performed as previously described with only minor modifications (12,19,44). Fig.…”

Section: Methodsmentioning

confidence: 99%

“…This sperm-DNA complex is then injected into the cytoplasm of MII-arrested oocytes (oocytes injected, oi), and the transgene is incorporated into the embryonic genome in a passive fashion via the endogenous DNA repair mechanisms (4). As previously observed for PNI, many transgenes integrate as multiple concatamerized vector copies (6,8,11,12). ICSI-Tr, originally developed to generate transgenic mice, has been extended with some success to other species (13).…”

mentioning

confidence: 99%

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

Marh

Stoytcheva

Urschitz

et al. 2012

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

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We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.transgene | genetically modified

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“…The procedures were performed as previously described with only minor modifications (12,19,44). Fig.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Marh

Stoytcheva

Urschitz

et al. 2012

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

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“…In line with this, Kaneko et al (2005) observed that all transgenic mice produced by RecA-ICSI were fully transgenic with all of them passing the trait to the next generation. Suganuma et al (2005) observed similar facilitated transgene insertion during a transposase enzyme-mediated insertion of a transposon during ICSI microinjections as a complex named transposome (TN:ICSI). However, Moreira et al (2007) found that although the transgenesis efficiency of the common ICSIbased transgenesis procedure with frozen-thawed sperm cells is similar to that of ICSI with fresh sperm cells and RecA-DNA complexes, the traditional ICSI-Tr procedure with frozen-thawed spermatozoa is much more efficient for maintaining a low frequency of founder animal mosaicism.…”

Section: Recombinase-mediated Pig Transgenesismentioning

confidence: 96%

“…In the mouse, both the bacterial recombinase protein recombinase-A (RecA; Kaneko et al 2005, Moreira et al 2007) and a mutated hyperactive Tn5 transposase protein (*Tn5p; Suganuma et al 2005) increase transgenesis several fold as compared with conventional methods such as pronuclear microinjection (Nakanishi et al 2002) and traditional ICSI-Tr (Perry et al 1999). The RecA protein from Escherichia coli is one of the best characterized of the recombinases, and plays an important role in homologous recombination and DNA repair in E. coli (Kowalczykowski & Eggleston 1994, Shinohara & Ogawa 1995.…”

Section: Introductionmentioning

confidence: 99%

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

García–Vázquez¹,

Ruiz²,

Matás³

et al. 2010

Reproduction

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Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) proteincoated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 mg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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“…ated gene transfer has been used to produce transgenic mice [15][16][17], rats [18] and pigs [13]. However, the production efficiency of transgenic pigs by ICSI-mediated gene transfer is low, and the likelihood of producing transgenic fetuses and live piglets is less than 1% [13].…”

mentioning

confidence: 99%

Effects of Sperm Pretreatment on Efficiency of ICSI-Mediated Gene Transfer in Pigs

Kurome

Saitô

Tomii

et al. 2007

Journal of Reproduction and Development

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Abstract. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.

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Tn5 Transposase-Mediated Mouse Transgenesis1

Cited by 39 publications

References 40 publications

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

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

Production of transgenic piglets using ICSI–sperm-mediated gene transfer in combination with recombinase RecA

Effects of Sperm Pretreatment on Efficiency of ICSI-Mediated Gene Transfer in Pigs

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