Sperm-mediated gene transfer: Production of pigs transgenic for a human regulator of complement activation

Lavitrano, Marialuisa; Forni, Monica; Varzi, Vincenzo; Pucci, L; Bacci, Maria Laura; Stefano, Carla Di; Fioretti, Daniela Piergili; Zoraqi, Grigor; Moioli, B.; Rossi, Massimo; Lazzereschi, Davide; Stoppacciaro, Antonella; Seren, E.; Alfani, D; Cortesini, Raffaello; Frati, Luigi

doi:10.1016/s0041-1345(97)00998-6

Cited by 66 publications

(42 citation statements)

References 10 publications

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“…Lavitrano et al (1989) described a new method for transgenic animal production: SMGT. This method is based on the ability of sperm to bind, internalize, and transport exogenous DNA into an oocyte during fertilization (Francolini et al 1993, Zani et al 1995, Lavitrano et al 1997. SMGT has been used more or less successfully in the production of transgenic embryos and animals in a large number of species (reviewed in Smith & Spadafora (2005)).…”

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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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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“…Subsequently, we successfully adapted and optimised the technique for use in large animals; in fact, it was highly efficient for the generation of human decay accelerating factor (hDAF) transgenic pig lines (Lavitrano et al 1997a(Lavitrano et al , 2002(Lavitrano et al , 2003, as well as multigene transgenic pigs, by simultaneously introducing three reporter genes, namely enhanced green fluorescent protein (EGFP), enhanced blue fluorescent protein (EBFP) and red fluorescent protein (DsRed2; Webster et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Sperm-mediated gene transfer

Lavitrano

Busnelli

Cerrito

et al. 2006

Reprod. Fertil. Dev.

115

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Abstract. Since 1989, a new method for the production of transgenic animals has been available, namely spermmediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

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“…Since the pioneering study by Brackett et al [1] showing that rabbit spermatozoa were able to incorporate SV40 DNA into their heads during incubation, the property of association of spermatozoa with heterologous DNA has been extensively studied [2][3][4][5]. These studies have facilitated the idea of using sperm as a vector to transfer exoge-actually carry the DNA into eggs, and the gene thus transferred is transcriptionally active [11][12][13][14][15][16][17]. In addition, as described in the associated paper [18], we have shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization (testis-mediated gene transfer).…”

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confidence: 99%

Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

Chang

Ikeda

Hayashi

et al. 1999

Journal of Reproduction and Development

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Abstract.We have recently shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization and expressed and transmitted in the descendants (testis-mediated gene transfer). In the present study, we investigated the association of epididymal and ejaculated sperm with DNA injected into the testis of rats. In the first experiment, a chimeric gene of mouse metallothionein-I promoter (MT)/human growth hormone receptor (hGHR) labeled with FITC was prepared. This gene construct was mixed with cationic liposome and injected into rat testis, and 4 days later the cauda epididymis was dissected out and prepared for histological examination. A confocal microscopic observation showed that not only the head, but also the tail, of almost all spermatozoa in the epididymis was labeled with FITC. In the next experiment, 4 days after the injection of MT/hGHR gene encapsulated by liposome into the testis, the male was allowed to mate with females and spermatozoa were recovered from the uterus in the next morning. Spermatozoa were incubated in the presence or absence of deoxyribonuclease (DNase) I and then genomic DNA was extracted. The MT/hGHR gene was detected only in the spermatozoa incubated without DNase by means of PCR. These results support the notion that, in the case of our testis-mediated gene transfer, exogenous DNA injected with liposomes is not integrated into genome of the sperm and the integration occurs after fertilization.

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Sperm-mediated gene transfer: Production of pigs transgenic for a human regulator of complement activation

Cited by 66 publications

References 10 publications

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

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

Sperm-mediated gene transfer

Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

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