Development of efficient strategies for the production of genetically modified pigs

Nagashima, Hiroshi; Fujimura, Tadao; Takahagi, Yoichi; Kurome, Mayuko; Wako, Naohiro; Ochiai, Takashi; Esaki, Ritsuko; Kano, Koichiro; Saito, Shoji; Okabe, Masaru; Murakami, Hiroshi

doi:10.1016/s0093-691x(02)01261-x

Cited by 56 publications

(57 citation statements)

References 35 publications

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“…There are some limitations for cloning technique, such as the need of several good quality embryos to produce and maintain a pregnancy [1]. Furthermore, porcine oocytes and embryos Table 4.…”

Section: Discussionmentioning

confidence: 99%

“…Total cell number Apoptotic cell number IAP (Index of Apoptosis) Total cell number Apoptotic cell number IAP (Index of Apoptosis) are even more sensitive to temperature fluctuations than those from other domestic species [1]. It is believed that for a successful cloning, the pattern of epigenetic modifications in the donor cells must be remodeled to be similar to the pattern present in the fertilized embryos [35].As a result, we performed embryo aggregation and obtained a significant increase in in vitro embryo development and cell number, 20% cleavage and a three-fold increase in blastocyst rate.…”

Section: Cloned 1x Blastocystmentioning

confidence: 99%

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18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

Buemo

Gambini

Moro

et al. 2016

Reprod. Fertil. Dev.

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In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

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Section: Discussionmentioning

confidence: 99%

Section: Cloned 1x Blastocystmentioning

confidence: 99%

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

Buemo

Gambini

Moro

et al. 2016

Reprod. Fertil. Dev.

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“…Also important are the efficacies of the exogenous somatic nuclei transplantation procedure for maternal chromosome elimination (recipient oocyte enucleation), the oocyte reconstruction technique and the clonal cybrid (cytoplasmic hybrid) activation system (Kato et al, 2000;Polejaeva et al, 2000;Wakayama and Yanagimachi, 2001;Galli et al, 2002;Roh and Hwang, 2002;Yin et al, 2002;Nagashima et al, 2003;Samiec et al, 2003a,b). At present, the most common embryo reconstruction techniques are nuclear transfer followed by electric pulse fusion of cytoplast (ooplast) with somatic cell (cattle: Cibelli et al, 1998;Wells et al, 1999;Kato et al, 2000;sheep: Wilmut et al, 1997;McCreath et al, 2000;goats: Baguisi et al, 1999;pigs: Koo et al, 2000;Polejaeva et al, 2000;Boquest et al, 2002;Yin et al, 2002) or the use of a suspension of inactivated Sendai virus (HVJ) particles with an accurately defined number of haemagglutination activity units (mice: Kato et al, 1999;Ono et al, 2001).…”

Section: Somatic Cloning In Various Species Of Mammalsmentioning

confidence: 99%

“…That is why in the experiments by Kano (2002) and Nagashima et al (2003), the pre-adipocyte cell lines derived from dedifferentiated mature adipocytes of the subcutaneous adipose tissue of adult pigs have recently been tested for nuclear transfer. Cell cycle synchronization at G0/G1 stages by differentiation induction with a combined isobutyl-methylxanthine, dexamethasone, insulin treatment is unique to the progenitor cells of adipocytes.…”

Section: Coordination Between Donor Cell Type and Cell Cycle Stage -Ementioning

confidence: 99%

Development of pig cloning studies: past, present and future

Samiec¹

2004

J. Anim. Feed Sci.

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The birth of the first nuclear transfer-derived cloned piglet resulted from the transfer of a nucleus from a 4-cell stage embryo to an enucleated oocyte. Although biotechnological procedures in swine have recently undergone tremendous progress, the efficiency of pig somatic cloning is still lower than in other species of farm animals and, when expressed as a proportion of reconstructed oocytes, it does not exceed an average of 5 to 10% blastocysts and 1.5% born piglets. The basic prerequisite for practical application of this method is to improve efficiency. This requires further detailed studies. Development of pig somatic cell cloning was inspired not only by the necessity for quick improvement of the efficiency of the nuclear transfer technique in this species, but above all by its possible practical application for multiplication of transgenic piglets, for use in transplantation medicine and immunology, also pharmacy and animal breeding. A deficit of organs for human allotransplantation became a stimulus to the search for new, alternative sources of grafts. Therefore, a particularly attractive aspect of pig somatic cloning is the possibility of generating large numbers of transgenic pig organs for xenotransplantation. Compatibility with the human ones in size, anatomy, and physiology makes this an attractive proposition.

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“…Porcine intracytoplasmic sperm injection (ICSI) to produce transgenic pigs has become indispensable to biomedical research. However, the rate of successful incorporation of the exogenous genes into the host genome is extremely low in animals [1] . Moreover, exogenous DNA injection into the ooplasm does not result in transgenic progeny [2] .…”

Section: Introductionmentioning

confidence: 99%

Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

Wu¹,

Chen²,

Wang³

et al. 2015

Front. Agr. Sci. Eng.

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Several reliable methods to produce transgenic animals use the male genome. After penetration into oocytes, sperm DNA undergoes dramatic conformational changes that might represent an opportunity for exogenous DNA to integrate into the zygote genome. A nuclease, DNase I, with Ca 2+ /Mg 2+ dependent activity and Zn 2+ inhibition, is one of the enzymes responsible for sperm DNA remodeling. To date, the effect of different calcium concentrations in manipulation media on porcine intracytoplasmic sperm injection has not been fully investigated. The present study was conducted to examine the effect of calcium in the surrounding media, and we found that the number of embryos expressing green fluorescent protein (EGFP) was increased in media containing Ca 2+ . However, the number did not change over Ca 2+ concentrations from 2 to 8 mmol$L -1 (P > 0.05). Moreover, free Ca 2+ concentrations in the media were found to affect the efficiency which is porcine intracytoplasmic sperm injection (ICSI) embryos expressing EGFP protein, which was related to the activation of ooplasmic DNase I. These findings reveal a mechanism and pathway involving EGFP expression in ICSI embryos.

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Development of efficient strategies for the production of genetically modified pigs

Cited by 56 publications

References 35 publications

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

Development of pig cloning studies: past, present and future

Effect of calcium on porcine ICSI embryos expressing EGFP is related to activation of ooplasmic DNase I

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