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.

Production of goats by somatic cell nuclear transfer

https://www.cell.com/current-biology/pdf/S0960-9822(01)00459-6.pdf

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells

Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

https://science.sciencemag.org/content/sci/289/5482/1188.full.pdf

Pig cloning by microinjection of fetal fibroblast nuclei

Cloning of mammals by nuclear transfer (NT) results in gestational or neonatal failure with at most a few percent of manipulated embryos resulting in live births. Many of those that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Cloned embryos derived from donors, such as embryonic stem cells, that may require little or no reprogramming of early developmental genes develop substantially better beyond implantation than NT clones derived from somatic cells. Although recent experiments have demonstrated normal reprogramming of telomere length and X chromosome inactivation, epigenetic information established during gametogenesis, such as gametic imprints, cannot be restored after nuclear transfer. Survival of cloned animals to birth and beyond, despite substantial transcriptional dysregulation, is consistent with mammalian development being rather tolerant to epigenetic abnormalities, with lethality resulting only beyond a threshold of faulty gene reprogramming encompassing multiple loci.

https://science.sciencemag.org/content/sci/293/5532/1093.full.pdf

Nuclear Cloning and Epigenetic Reprogramming of the Genome

Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.

https://science.sciencemag.org/content/sci/282/5396/2095.full.pdf

Eight Calves Cloned from Somatic Cells of a Single Adult

Twenty-four calves were cloned from six somatic cell types of female and male adult, newborn and fetal cows. The clones were derived from female cumulus (n = 3), oviduct (n = 2) and uterine (n = 2) cells, female and male skin cells (n = 10), and male ear (n = 5) and liver (n = 2) cells. On the basis of the number of cloned embryos transferred (n = 172) to surrogate cows, the overall rate of success was 14%, but based on the number of surrogate mothers that became pregnant (n = 50), the success rate was 48%. Cell nuclei from uterus, ear and liver cells, which have not been tested previously, developed into newborn calves after nuclear transfer into enucleated oocytes. To date, seven female and six male calves have survived: six of the females were from adult cells (cumulus (n = 3), oviduct (n = 2) and skin (n = 1) cells) and one was from newborn skin cells, whereas the male calves were derived from adult ear cells (n = 3), newborn liver and skin cells (n = 2), and fetal cells (n = 1). Clones derived from adult cells frequently aborted in the later stages of pregnancy and calves developing to term showed a higher number of abnormalities than did those derived from newborn or fetal cells. The telomeric DNA lengths in the ear cells of three male calves cloned from the ear cells of a bull aged 10 years were similar to those of the original bull. However, the telomeric DNA lengths from the white blood cells of the clones, although similar to those in an age-matched control, were shorter than those of the original bull, which indicates that telomeric shortening varies among tissues.

/pdf/cloning-of-calves-from-various-somatic-cell-types-of-male-2u6v6zeylw.pdf

Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows.

PROBLEM TO BE SOLVED: To elucidate and provide a somatic nucleus initializing factor useful in creating somatic clone. SOLUTION: The somatic nucleus initializing factor is a phosphorylated acidic protein derived from the cytoplasm of bovine unfertilized embryo and having a molecular weight of 20-25 KDa determined by two-dimensional electrophoresis using a protein marker, particularly a protein having combinedly with a peptide chain having a specific amino acid sequence. COPYRIGHT: (C)2004,JPO

Somatic nucleus initializing factor

PROBLEM TO BE SOLVED: To use bean curd refuse higher in nutritive value than Tofu (bean curd) but with nearly all thereof destined to be an industrial waste, as a food for humans by 100% without being used as a fertilizer or feed, thus greatly contributing to the utilization of soybean resource without giving any industrial waste, air pollution control and environmental cleaning with no incineration disposal, no-wasting campaign, etc. SOLUTION: Bean curd refuse as byproduct in the process of producing Tofu is subjected to salting treatment and stored at room temperature; thereafter, together with soy residues, is made into a food by 100% as Miso (fermented soybean paste) or its compound to be reused; thus no industrial wastes including such bean curd refuse and soy sauce refuse are produced. COPYRIGHT: (C)2011,JPO&INPIT

Yukio Tsunoda

Papers

Eight Calves Cloned from Somatic Cells of a Single Adult

Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows.

Somatic nucleus initializing factor

Method for storing and utilizing bean-curd refuse with nearly all thereof destined to be industrial waste