Nuclear Transfer with Germ Cells

2014 ◽  
pp. 53-62
Author(s):  
Jiyoung Lee ◽  
Takashi Kohda ◽  
Fumitoshi Ishino
Keyword(s):  
Germ Cells ◽  
Nuclear Transfer

208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

2006 ◽  
Vol 18 (2) ◽  
pp. 212
Author(s):  
J. Y. Won ◽  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. H. Kang ◽  
H. Shim
Keyword(s):  
Human Serum ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
In Vitro Model ◽  
Regulatory Protein ◽  
Cell Types ◽  
Human Complement ◽  
Transgenic Pigs ◽  
Vitro Model

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

scholarly journals Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

1996 ◽  
Vol 46 (5) ◽  
pp. 871-880 ◽  
Author(s):  
A. Moens ◽  
P. Chesné ◽  
F. Delhaise ◽  
A. Delval ◽  
F-J. Ectors ◽  
...  
Keyword(s):  
Germ Cells ◽  
Nuclear Transfer ◽  
Fetal Bovine

scholarly journals The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

2015 ◽  
Vol 38 (2) ◽  
pp. 220-226 ◽  
Author(s):  
Vahid Mansouri ◽  
Mohammad Salehi ◽  
Mohsen Nourozian ◽  
Fatemeh Fadaei ◽  
Reza Mastery Farahani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Embryonic Stem

Microinsemination and Nuclear Transfer Using Male Germ Cells

2005 ◽  
pp. 189-229 ◽  
Author(s):  
Atsuo Ogura ◽  
Narumi Ogonuki ◽  
Hiromi Miki ◽  
Kimiko Inoue
Keyword(s):  
Germ Cells ◽  
Nuclear Transfer ◽  
Male Germ Cells

Germ cell nuclei of male fetal mice can support development of chimeras to midgestation following serial transplantation

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 779-783 ◽  
Author(s):  
Y. Kato ◽  
Y. Tsunoda
Keyword(s):  
Germ Cells ◽  
Nuclear Transfer ◽  
Yolk Sac ◽  
Glucose Phosphate Isomerase ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Glucose Phosphate ◽  
Fetal Mice ◽  
Serial Nuclear Transfer

Chimeric embryos between fertilized eggs from F1 (C57BL × CBA) and 15.5-16.5 days post coitum (dpc) male fetal germ cells (FGCs) from CD-1 strain (glucose phosphate isomerase, Gpi-1a/a) mice were produced by nuclear transfer. Briefly, a single FGC was fused with enucleated oocytes and activated, and the reconstituted oocytes were cultured to the 2-cell stage. The nucleus from the reconstituted 2-cell embryos was then transferred into an enucleated blastomere of the same stage embryos derived from F1 mice to produce chimeric embryos. The reconstituted 2-cell embryos, which synchronously divided to the 4-cell stage after treatment with nocodazole, were further cultured in vitro. Compacted morula and blastocysts were transferred to the uteri of pseudopregnant female mice. Some recipients were allowed to develop to term and the others were killed at mid gestation to analyze the contribution of donor FGC-derived cells. Survival to term was low with no chimeric animals. Glucose phosphate isomerase (GPI) analysis at midgestation revealed that some conceptuses had chimerism in the fetuses, trophoblast and yolk sac at day 10.5 of pregnancy. The contribution of donor cells was 37–47%, 19–65% and 12–63%, respectively. It was concluded that the nucleus from 15.5-16.5 dpc male fetal germ cells had the potency to develop into fetus, trophoblast and yolk sac after serial nuclear transfer with oocytes and fertilized embryos. The reason for the low viability of chimeric embryos is discussed.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Susa Shin ◽  
Kwang Sung Ahn ◽  
Seong-Jun Choi ◽  
Soon Young Heo ◽  
Hosup Shim
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Neural Precursor ◽  
Blastocyst Development ◽  
Donor Cells

SummaryUndifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.

23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 130
Author(s):  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. Y. Won ◽  
H. Shim
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Somatic Cells ◽  
Dna Demethylation ◽  
Term Survival ◽  
Cpg Sites ◽  
Fetal Fibroblasts

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P &lt; 0.05), and DNA demethylation occurred in embryos from both EGCNT (P &lt; 0.05) and SCNT (P &lt; 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P &lt; 0.01) and ICSI (P &lt; 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.

Microinsemination and Nuclear Transfer with Male Germ Cells

2002 ◽  
pp. 175-186
Author(s):  
Atsuo Ogura ◽  
Narumi Ogonuki ◽  
Kimiko Inoue
Keyword(s):  
Germ Cells ◽  
Nuclear Transfer ◽  
Male Germ Cells
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