A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Pereyra-Bonnet, F.; Fernández-Martı́n, Rafael; Olivera, R.; Jarazo, Javier; Vichera, G.; Gibbons, Ann; Salamone, D.

doi:10.1071/rd07172

Cited by 45 publications

(38 citation statements)

References 63 publications

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“…Another innovation in SMGT has been the use of ICSI to deliver transgene-containing sperm cells directly into the egg, a process known as 'ICSI-mediated transgenesis' (ICSI-Tr) which was reported for the first time in mice (Perry et al 1999) and later developed in pigs , Pereyra-Bonnet et al 2008, García-Vázquez et al 2009, Wu et al 2009). These techniques (pronuclear injection and ICSI) are considered as passive methods for transgenesis (Shinohara et al 2007), which rely on the repair mechanisms of the host for transgene insertion.…”

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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“…In the present study, we were able to successfully collect sperm from collared peccary testis xenografts and, also for the first time, harvest peccary oocytes, which subsequently after standard ICSI procedures (Pereyra-Bonnet et al 2008) resulted in diploid embryos. Using the PCR technique, we observed that twothirds of the four-cell embryos obtained expressed the paternally imprinted gene NNAT (Park et al 2011, Chankitisakul et al 2012.…”

Section: Experimental Groupmentioning

confidence: 80%

“…As the number of obtained viable oocytes was small, ICSI procedure was carried out using spermatozoa only from testis cell suspension xenografts (eight oocytes), and parthenogenetic embryos were also produced as a control (Pereyra-Bonnet et al 2008). Embryo rate was assessed 24 and 48 h after ICSI, and the DNA content of the parthenogenetic and ICSI embryos was evaluated using NanoDrop (ND1000, Thermo Scientific).…”

Section: Oocyte Sampling and In Vitro Maturation And Icsimentioning

confidence: 99%

Derivation of sperm from xenografted testis cells and tissues of the peccary (Tayassu tajacu)

Campos-Júnior¹,

Costa²,

Avelar³

et al. 2014

Reproduction

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Because the collared peccary (Tayassu tajacu) has a peculiar Leydig cell cytoarchitecture, this species represents a unique mammalian model for investigating testis function. Taking advantage of the well-established and very useful testis xenograft technique, in the present study, testis tissue and testis cell suspensions from immature collared peccaries (nZ4; 3 months old) were xenografted in SCID mice (nZ48) and evaluated at 2, 4, 6, and 8 months after grafting. Complete spermatogenesis was observed at 6 and 8 months after testis tissue xenografting. However, probably due to de novo testis morphogenesis and low androgen secretion, functionally evaluated by the seminal vesicle weight, a delay in spermatogenesis progression was observed in the testis cell suspension xenografts, with the production of fertile sperm only at 8 months after grafting. Importantly, demonstrating that the peculiar testicular cytoarchitecture of the collared peccary is intrinsically programmed, the unique Leydig cell arrangement observed in this species was re-established after de novo testis morphogenesis. The sperm collected from the xenografts resulted in diploid embryos that expressed the paternally imprinted gene NNAT after ICSI. The present study is the first to demonstrate complete spermatogenesis with the production of fertile sperm from testis cell suspension xenografts in a wild mammalian species. Therefore, due to its unique testicular cytoarchitecture, xenograft techniques, particularly testis cell suspensions, may represent a new and very promising approach to evaluate testis morphogenesis and to investigate spermatogonial stem cell physiology and niche in the collared peccary.

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“…The number of good quality blastocysts produced following ICSI in our experiments approached those produced by normal IVF. Although further work might push the technology further, the number and quality of embryos obtained with our approach could boost the use of ICSI in several fields of reproduction biotechnologies, such as the production of large transgenic animals as disease models [22,23], as well as the replacement/expansion of animals threatened with extinction [10].…”

Section: Discussionmentioning

confidence: 99%

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

Anzalone

Iuso

Czernik

et al. 2016

J Assist Reprod Genet

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Purpose This study aims to determine if the integrity of the sperm plasma membrane and acrosome vesicle could be limiting factors in sheep intracytoplasmic sperm injection (ICSI). Methods Prior to in vitro fertilization (IVF) or ICSI, the oocytes were subjected to in vitro maturation (IVM) for 24 h. First, to evaluate the need of artificial activation for ovine ICSI, 226 oocytes were injected with intact spermatozoa (IS), from which 125 were activated by incubation in ionomycin and 101 were cultured without activation. Next, spermatozoa were mechanically (by piezo-electrical pulses) and/or chemically (by ionomycin/Triton X-100) treated to break membranes and acrosomes and were injected into oocytes, grouped as follows: (i) piezo-pulsed spermatozoa (PPS), (ii) PPS pre-treated with ionomycin (PPS-I), (iii) PPS pre-treated with Triton X-100 (PPS-T), and (iv) intact and untreated spermatozoa as a control (CTR-IS). Results No differences were observed in the zygote/cleavage/ blastocyst rate between chemically activated and nonactivated oocytes (50 vs. 45 %, 11.6 vs. 10.1 %; 1.8 vs. 1.1 %, respectively), after ICSI with CTR-IS. Injection of PPS compared to CTR-IS increased the proportion of zygotes and blastocysts (84.6 vs. 45 %, p < 0.01; 15.5 vs. 1.1 %, p < 0.0001, respectively). Moreover, the percentage of PPSderived blastocysts was not significantly different from that obtained by conventional IVF (15.5 vs. 20.2 %). The ICSI blastocysts' development was also improved with PPS pretreated with ionomycin (15.6 %), but was completely impeded with PPS pre-treated with Triton X-100 (0 %). Conclusion Our findings confirm that ICSI with spermatozoa whose plasma membrane and acrosome have been mechanically damaged substantially improves embryonic development until the blastocyst stage.

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A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species

Cited by 45 publications

References 63 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

Derivation of sperm from xenografted testis cells and tissues of the peccary (Tayassu tajacu)

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

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