Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Kurome, Mayuko; Ishikawa, Takayuki; Tomii, Ryo; Ueno, Satoshi; Shimada, Arata; Yazawa, Hajime; Nagashima, Hiroshi

doi:10.1262/jrd.19165

Cited by 55 publications

(50 citation statements)

References 55 publications

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“…The different interpretations may have resulted from the variations in species, strains or cell types used in the respective studies. (36). In the present study, the average number of blastocyst cells was significantly lower for the embryos reconstructed using transfected fibroblasts.…”

Section: Discussioncontrasting

confidence: 44%

Comparative analysis of various donor cell types for somatic cell nuclear transfer and its association with apoptosis and senescence

Kim

Hyun

2013

Molecular Medicine Reports

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Abstract. The aim of the present study was to characterize potential somatic cell nuclear transfer (SCNT) donor cells by comparing two lines of transfected cells with their non-modified parental controls in culture. Fetal fibroblasts used in the study originated from crossbred Landrace x Yorkshire x Duroc (LYD) or Yucatan mini-pigs. The LYD fibroblasts were modified by the transfection of a tetracycline on/off gene, whereas Yucatan fibroblasts were triple transfected with the complement regulatory factors, human decay-accelerating factor and human CD59, as well as H-transferase. At the 9th doubling passage, parameters associated with senescence and apoptosis, including morphology, mRNA expression (TP53, Bcl-2, Bax) and reactive oxygen species (ROS) levels, were evaluated. Population doubling (PD) time was calculated by assessing the time required for cell numbers to double by averaging the three cell passages. Quantitative polymerase chain reaction revealed that when comparing LYD with Yucatan fibroblasts, the latter exhibited a lower relative expression of TP53 and a higher relative expression of antiproliferative Bcl-2, which correlated with the PD time results (26 and 40 h, respectively). Tetracycline on/off transfected cell lines exhibited a lower relative expression of antiapoptotic Bcl-2 compared with their originating LYD cells. Similarly, triple transgenic cells exhibited higher TP53 and Bax mRNA expression levels than their non-transgenic counterparts. For ROS measurement, cells were incubated with 2',7'-dichlorofluorescin diacetate under the same conditions and were analyzed by flow cytometry. Yucatan fibroblasts exhibited higher ROS content than LYD cells. In addition, the two transgenic cell lines produced higher ROS levels than their corresponding non-transfected cell lines. In conclusion, these results indicate that characteristics associated with senescence and apoptosis in transfected cells during culture may affect the efficiency of SCNT when used as donor cells for the production of transgenic swine.

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Section: Discussioncontrasting

confidence: 44%

Comparative analysis of various donor cell types for somatic cell nuclear transfer and its association with apoptosis and senescence

Kim

Hyun

2013

Molecular Medicine Reports

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“…In pigs, a signal from three or more embryos is required to maintain pregnancy (King et al, 2002). The pregnancy rate and delivery rate of surrogate females receiving cloned embryos is usually lower than that resulting from transfer of unmanipulated embryos (Hornen et al, 2007;Kurome et al, 2008;Lagutina et al, 2007;Park et al, 2010). The major possible reason for this is that the low developmental ability of cloned embryos significantly reduces their signaling to the recipient mother after embryo transfer, and thus they are unable to cause the recipients establish pregnancy or maintain pregnancy to term.…”

Section: Discussionmentioning

confidence: 99%

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

Shi

Liu

et al. 2013

Cellular Reprogramming

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Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones ( = total number of healthy cloned piglets born / total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 -0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 -0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 -0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provi...

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“…The difference in strain of the surrogate miniature pig affects the post-implantation development of miniature pig NT embryos [34]. On the other hand, viable cloned [30,35] and transgenic [36] miniature pigs have been produced using domestic pig oocytes as recipient oocytes and surrogate mothers.…”

Section: Discussionmentioning

confidence: 99%

“…The usefulness of miniature pigs as an animal model has been proven; but nevertheless, only a limited number of studies have reported on cloned miniature pigs derived from somatic donor cells [30,31,[34][35][36]. Because of the comparison with domestic pigs, production of cloned miniature pigs using miniature pigs oocytes has limitations.…”

Section: Lee Et Al 260mentioning

confidence: 99%

“…These findings are similar to the observations of the above-mentioned studies. Our group has observed the expression profiles of genes involved in transcription, DNA methylation, histone deacetylation, apoptosis and embryonic growth at different developmental stages, and some genes in embryos cloned with MSCs closely resembled those of in vivo produced embryos [7].The usefulness of miniature pigs as an animal model has been proven; but nevertheless, only a limited number of studies have reported on cloned miniature pigs derived from somatic donor cells [30,31,[34][35][36]. Because of the comparison with domestic pigs, production of cloned miniature pigs using miniature pigs oocytes has limitations.…”

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confidence: 99%

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Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

Lee

Kang

Maeng

et al. 2010

Journal of Reproduction and Development

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Abstract. The present study compared the developmental ability of miniature pig embryos cloned with fetal fibroblasts (FFs), bone marrow-derived mesenchymal stem cells (MSCs) and differentiated (osteocytes, adipocytes and chondrocytes) MSCs. MSCs were isolated from an approximately 1-month-old female miniature pig (T-type, PWG Micro-pig ® , PWG Genetics Korea). MSCs were differentiated into osteocytes, adipocytes and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, as well as FFs of miniature pig, were transferred into enucleated oocytes of domestic pigs. Data from 10 replicates involving 1567 cloned embryos were assessed in terms of developmental rates. The in vitro development rate to the blastocyst stage of embryos cloned with undifferentiated MSCs was significantly (P<0.05) higher than that of embryos cloned with differentiated MSCs and FFs. Surgical transfer of 523 two-cell stage embryos cloned with undifferentiated MSCs into five synchronized domestic pig recipients resulted in 5 cloned miniature pig offspring (1 stillborn and 4 viable) from 2 pregnant recipients. The results imply that MSCs might be multipotent and that they can be used to produce viable cloned miniature pigs that cannot be easily reproduced with differentiated somatic cells. Key words: Animal cloning, Differentiation, Mesenchymal stem cells, Miniature pig, Nuclear transfer (J. Reprod. Dev. 56: [256][257][258][259][260][261][262] 2010) he production of cloned animals by nuclear transfer (NT) has been increasingly studied as a potential approach to tissue and organ xenotransplantation with reduced immunogenicity for endstage organ failure. Several studies have suggested that miniature pigs, rather than nonhuman-primate species, are suitable as a donor animal for human xenotransplantation [1,2] because miniature pigs are superior in terms of similarities in gross anatomy and physiology to humans as well as in terms of ethical issues. The NT efficiency, however, remains relatively low due to abnormalities throughout pre-and post-implantation development regardless of the species or type of donor cell [3]. A number of factors affect NT efficiency, including the NT technical process, activation protocol, recipient oocyte age, cell cycle stage and type of donor cells, which has been implicated in the precise reprogramming of chromatin and genomic imprinting. Among these factors, incomplete selection of donor cells has also been considered a prime cause for NT inefficiency.Transfer of embryonic stem (ES) cells to produce cloned embryos has resulted in consistently higher numbers of viable offspring compared with somatic cells in mice [4,5]. Moreover, embryos cloned with porcine mesenchymal stem cells (MSCs) and their derivatives along the osteogenic lineage give rise to an increase in the rate of preimplantation development compared with adult fibroblasts [6]. Our previous study on gene expression profiles demonstrated that some ge...

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Production of Transgenic and Non-Transgenic Clones in Miniature Pigs by Somatic Cell Nuclear Transfer

Cited by 55 publications

References 55 publications

Comparative analysis of various donor cell types for somatic cell nuclear transfer and its association with apoptosis and senescence

Comparative analysis of various donor cell types for somatic cell nuclear transfer and its association with apoptosis and senescence

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

Developmental Ability of Miniature Pig Embryos Cloned with Mesenchymal Stem Cells

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