Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes

In contrast to nuclear DNA, cytoplasmic genes may differ among cloned animals due to the presence of polymorphic mitochondrial DNA haplotypes in the host oocytes, raising doubts about histocompatibility among clones. Three bovine clones were generated by nuclear transfer; dermal fibroblasts from a fetus were used as donor cells, whereas oocytes from abbatoir-derived ovaries were used as recipient cells. The mitochondrial DNA (sequencing of coding and non-coding regions) and nuclear DNA (13 microsatellite markers) of cloned and control animals were characterized to identify potential polymorphisms. Skin auto- and allografts were transplanted on the adult clones and a non-related animal as a measure of immunological reactivity. Nuclear DNA of cloned animals was genetically identical but differed in all microsatellites of the non-related control. Amounts of donor cell mitochondrial DNA in the skin ranged from 1 to 2.6% among clones. Few differences in heteroplasmy were observed between skin and WBC of the clones, indicating limited mitochondrial DNA segregation in tissues during pre- and post-natal development to adulthood. Sequencing of the remaining oocyte-derived mitochondrial DNA haplotype identified polymorphisms in coding and non-coding regions, confirming their origin from unrelated maternal lineages. Nonetheless, skin transplants between clones were accepted for the 92 d study period, whereas third-party grafts were rejected. In conclusion, the nuclear transfer-generated adult bovine clones used in this study were immunologically compatible with one another despite differences in their mitochondrial DNA haplotypes.     

Dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs

Pigs have been recognized as an excellent biomedical model for investigating a variety of human health issues. We developed genetically modified pigs that exhibit the apparent symptoms of diabetes. Transgenic cloned pigs carrying a mutant human hepatocyte nuclear factor 1α gene, which is known to cause the type 3 form of maturity-onset diabetes of the young, were produced using a combined technology of intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. Although most of the 22 cloned offspring obtained died before weaning, four pigs that lived for 20–196 days were diagnosed as diabetes mellitus with nonfasting blood glucose levels greater than 200 mg/dl. Oral glucose tolerance test on a cloned pig also revealed a significant increase of blood glucose level after glucose loading. Histochemical analysis of pancreas tissue from the cloned pigs showed small and irregularly formed Langerhans Islets, in which poor insulin secretion was detected.     

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues.     

Cloned zebrafish by nuclear transfer from long-term-cultured cells

Although mammals have been cloned from genetically manipulated cultured cells, a comparable achievement has not been realized in lower vertebrates. Here we report that fertile transgenic zebrafish can be obtained by nuclear transfer using embryonic fibroblast cells from long-term cultures. The donor nuclei, modified by retroviral insertions expressing green fluorescent protein (GFP), were transplanted into manually enucleated eggs. Overall, a 2% success rate was achieved, resulting in 11 adult transgenic zebrafish expressing GFP. These nuclear transplants produced fertile, diploid offspring, and their F1/F2 progeny continued to express GFP in a pattern identical to that of the founder fish. This finding demonstrates that slowly dividing nuclei from cultured cells can be reprogrammed to support rapid embryonic development and sets up a foundation for targeted genetic manipulation in zebrafish.     

Recent progress and problems in animal cloning

It is remarkable that mammalian somatic cell nuclei can form whole individuals if they are transferred to enucleated oocytes. Advancements in nuclear transfer technology can now be applied for genetic improvement and increase of farm animals, rescue of endangered species, and assisted reproduction and tissue engineering in humans. Since July 1998, more than 200 calves have been produced by nuclear transfer of somatic cell nuclei in Japan, but half of them were stillborn or died within several months of parturition. Morphologic abnormalities have also been observed in cloned calves and embryonic stem cell-derived mice. In this review, we discuss the present situation and problems with animal cloning and the possibility for its application to human medicine.     

Survival of skin allografts following embryonic limb bud transplants in the turtle,Chelydra serpentina

Allografts of skin were observed in Chelydra serpentina. The response to these grafts was modified by a previous transplantation of a limb bud at an early embryonic stage. When the same donor was used for all transplants, the first skin graft was accepted by the host. A second skin graft, however, was rejected at about the rate of a simple first set allograft of skin. The animals were conditioned by the embryonic limb graft; this embryonic graft can be undergoing rejection at the same time a first set skin graft from the same donor was being accepted. The tolerance induced by the embryonic graft was sepcific for its donor.     

Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar

Cloned pigs were produced from cultured skin fibroblasts derived from a H-transferase transgenic boar. One 90 day fetus and two healthy piglets resulted from nuclear transfer by fusion of cultured fibroblasts with enucleated oocytes. The cells used in these studies were subjected to an extensive culture time, freezing and thawing, and clonal expansion from single cells prior to nuclear transfer. PCR and FACS analysis determined that the cloned offspring contained and expressed the H-transferase transgene. Microsatellite analysis confirmed that the clones were genetically identical to the boar. The cell culture and nuclear transfer procedures described here will be useful for applications requiring multiple genetic manipulations in the same animal.     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.     

Phenotypic characterization of Hanwoo (native Korean cattle) cloned from somatic cells of a single adult

We investigated phenotypic differences in Hanwoo cattle cloned from somatic cells of a single adult. Ten genetically identical Hanwoo were generated by somatic cell nuclear transfer from a single adult. Weights at birth, growing pattern, horn and noseprint patterns were characterized to investigate phenotypic differences. The weights of clones at 6 and 12 months were slightly heavier than that of the donor. A horn pattern analysis revealed that seven clones had exactly the same horn pattern as the donor cow, whereas three were different. Although similarities such as general appearance can often be used to identify individual cloned animals, no study has characterized noseprint patterns for this end. A noseprint pattern analysis of all surviving clones showed that all eight animals had distinct noseprints. Four were similar to the donor, and the remaining four had more secondary-like characteristics.     

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.     

Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer

The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2 % of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.     

Early and delayed aspects of nuclear reprogramming during cloning

The successful production of viable progeny following adult somatic cell nuclear transfer (cloning) provides exciting new opportunities for basic research for investigating early embryogenesis, for the propagation of valuable or endangered animals, for the production of genetically engineered animals, and possibly for developing therapeutically valuable stem cells. Successful cloning requires efficient reprogramming of gene expression to silence donor cell gene expression and activate an embryonic pattern of gene expression. Recent observations indicate that reprogramming may be initiated by early events that occur soon after nuclear transfer, but then continues as development progresses through cleavage and probably to gastrulation. Because reprogramming is slow and progressive, cloned embryos have dramatically altered characteristics in comparison with fertilized embryos. Events that occur early following nuclear transfer may be essential prerequisites for the later events. Additionally, the later reprogramming events may be inhibited by sub-optimum culture environments that exist because of the altered characteristics of cloned embryos. By addressing the unique requirements of cloned embryos, the entire process of reprogramming may be accelerated, thus increasing cloning efficiency.     

克隆猪技术及其在转基因猪研究中的应用

哺乳动物核移植技术是一种可以获得基因组遗传信息完全相同的后代的生物技术。猪体细胞核移植技术包括以下几个环节：卵母细胞的体外成熟、供体细胞的分离和处理、体细胞的核转移、重构胚胎的人工激活、胚胎体外培养和胚胎移植。由于该技术在最近几年的迅速发展,很多实验室已通过该技术成功获得了克隆猪后代。核移植克隆猪技术的出现为生产转基因猪提供了一种有效的方法,并且是目前生产基因打靶猪的惟一方法。至今利用克隆猪技术已经成功获得了一系列的转基因猪和基因敲除猪。以核移植技术产生基因修饰猪目前正处于从基础研究走向应用的过渡阶段。尽管猪体细胞核移植克隆的效率（出生克隆猪数占所用卵数的比例）还不高,但是由于通过该技术能够对猪基因组进行特定的修饰,确保生产的克隆动物100％为转基因动物,从而大大提高了转基因猪的制作效率,可以预料猪核移植技术将会对医药业和农业产生重大的影响。     

体细胞克隆牛产品安全分析

体细胞克隆技术是将已分化的体细胞移到去核的成熟卵母细胞中,通过体外激活和培养,再移植入受体母畜子宫内,繁殖出具有相同基因型后代的一种技术。该技术可以大幅提升繁殖效率,并提供高质、充足和营养丰富的动物食品。近年来,美国、日本和欧洲等国家相继宣布体细胞克隆动物食品可以上市。然而,目前体细胞克隆效率相当低下,即使是出生的克隆动物也往往伴随发育畸形或高死亡率等现象,在对克隆动物发育异常知之甚少的情况下,宣布克隆动物产品上市是否为时过早?以下综述了克隆牛肉、奶及其产品安全。     

Creating genetically modified pigs by using nuclear transfer

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture.     

Therapeutic cloning: concepts and practicalities

The concept of using embryonic stem (ES) cells as a source of multiple cell types for use in tissue repair has existed for approximately 20 years. Recent breakthroughs in somatic nuclear transfer and human ES cell derivation have produced a flurry of new activity in this area, with the recognition that ES cell lines that are customized and genetically identical to those of the patient are a distinct possibility. This article examines the background of and prospects for these exciting new developments.     

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.     

DNA甲基化和组蛋白修饰在克隆动物发育过程中的作用

体细胞核移植在农业应用、生产疾病模型动物、转基因家畜或产生人胚胎干细胞来治疗人类的疾病方面有巨大的应用潜力。虽然已经成功克隆出多种哺乳动物, 但该技术仍存在一些未解决的问题, 包括产生克隆动物的效率低和克隆动物的异常等。异常的表观遗传重编程是克隆胚胎发育失败的一个重要因素。文章重点论述了DNA甲基化、组蛋白修饰及其与克隆胚胎发育的关系。了解表观遗传调控机制有助于解决核移植技术中存在的问题, 有利于更好地应用这项技术。     

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.