Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

Lavitrano, Marialuisa; Stoppacciaro, Antonella; Bacci, Maria Laura; Forni, Monica; Fioretti, Daniela Piergili; Pucci, L; Stefano, Carla Di; Lazzereschi, Davide; Rughetti, Aurelia; Ceretta, S; Zannoni, Augusta; Rahimi, Hamid Reza; Moioli, B.; Rossi, Massimo; Nuti, Marianna; Rossi, Giancarlo; Seren, E.; Alfani, D; Cortesini, Raffaello; Frati, Luigi

doi:10.1016/s0041-1345(98)01863-6

Cited by 28 publications

(14 citation statements)

References 7 publications

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“…Establishment of the porcine IVM-ICSI procedure may help to generate transgenic pigs by sperm-mediated gene transfer. The possibility of mammalian spermatozoa being able to act as vectors for foreign DNA has been reported by Brackett et al [38], and the successful fertilization by gene-mediated spermatozoa has been reported in mice [39], sea urchins [40], and pigs [41][42][43]. This method, combined with ICSI, has succeeded in mice [34] and is now expected to be used in other mammalian species, including pigs.…”

Section: Discussionmentioning

confidence: 97%

Viable Piglets Generated from Porcine Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Head Injection

Nakai

Kashiwazaski

Takizawa

et al. 2003

Biology of Reproduction

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Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.

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Section: Discussionmentioning

confidence: 97%

Viable Piglets Generated from Porcine Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Head Injection

Nakai

Kashiwazaski

Takizawa

et al. 2003

Biology of Reproduction

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“…Several groups have successfully produced transgenic pigs using sperm as a vector for foreign genes (Lavitrano et al, 1997(Lavitrano et al, , 1999(Lavitrano et al, , 2002(Lavitrano et al, , 2003Lai et al, 2001;Chang et al, 2002). Each study was characterized by the specific method used to induce DNA-sperm binding and fertilize the oocytes.…”

Section: Discussionmentioning

confidence: 99%

“…Studies by Lai et al (2001) and Chang et al (2002) used DNA-liposome complexes or monoclonal antibodies to link foreign DNA to sperm, respectively. On the other hand, Lavitrano et al (1997Lavitrano et al ( , 1999Lavitrano et al ( , 2002Lavitrano et al ( , 2003 simply incubated sperm with DNA constructs for 2 h. In most of these cases (Lavitrano et al, 1997(Lavitrano et al, , 1999(Lavitrano et al, , 2002(Lavitrano et al, , 2003Chang et al, 2002), artificial insemination was used to get the DNA-carrying sperm to fertilize the oocytes.…”

Section: Discussionmentioning

confidence: 99%

Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

et al. 2006

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The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.

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“…The transgene was expressed for at least several months after the birth of the only transgenic calf. On the other hand, Lavitrano (Lavitrano et al, 1997, 1999 keeps on producing transgenic pigs with a very high rate of success (54% of the piglets born were transgenics) just by incubating DNA with spermatozoa before fertilization.…”

Section: Introductionmentioning

confidence: 99%

Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events

2000

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There are several methods of modifying bovine genomes. Pronuclear microinjection is more widely used but it is still to be improved. Searches for alternatives have lead to the development of new methods including SMGT (Sperm Mediated Gene Transfer), in which live spermatozoa are used as vehicles for DNA delivery during in vitro fertilization. In previous studies, we presented evidence that a highly repetitive Alu‐like repeat favours transgenesis by homologous recombination (HR). Up to 60% integration via HR was obtained following pronuclear microinjection of a Pst1 β‐actin GFP DNA construction. In the present study, we show that HR‐mediated integration is also possible using SMGT, since bovine spermatozoa electroporated with the same DNA construct are able to transfer it to a high proportion of embryos obtained by in vitro fertilization. Swim‐up selected bovine spermatozoa were mixed with the Pst1 β‐actin GFP construct (6 × 106 spermatozoids were incubated with 600 ng of μDNA), submitted or not to electroporation (300 V, 25 F) and treated or not with DNase I. The process of electroporation itself did not affect in vitro embryonic development. However, oocytes fertilized with electroporated DNA‐treated spermatozoa developed beyond the 16‐cell stage in proportions that were significantly lower (27% with Pst1 β‐actin GFP and 34% with β‐actin GFP) compared to the control without DNA (44%). On the other side, the use of electroporation significantly increased the uptake of DNA. The number of homologous recombination events detected by PCR went from 3.5% without electroporation to 46.5% after electroporation. In conclusion, our results confirm that spermatozoa electroporation combined with homologous recombination in a highly repetitive Pst1 sequence is a feasible method to obtain transgenic bovine embryos. Mol. Reprod. Dev. 57:338–345, 2000. © 2000 Wiley‐Liss, Inc.

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Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

Cited by 28 publications

References 7 publications

Viable Piglets Generated from Porcine Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Head Injection

Viable Piglets Generated from Porcine Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Head Injection

Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

Electroporation of bovine spermatozoa to carry DNA containing highly repetitive sequences into oocytes and detection of homologous recombination events

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