scholarly journals 34EFFECT OF GENOTYPE AND CELL LINE ON THE EFFICIENCY OF LIVE CALF PRODUCTION BY SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 139 ◽  
Author(s):  
K. DeLegge ◽  
M. Maserati ◽  
N. Kieser ◽  
D. Delanski ◽  
B. Henderson ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Early Adulthood ◽  
The Other ◽  
Culture Techniques ◽  
Clostridial Infection ◽  
Healthy Calf

The efficiency of production of live calves using somatic cell nuclear transfer was compared among 52 different cell lines representing 43 different genotypes. Cell lines were not genetically modified. Nuclear transfer was performed according to methods described by Cibelli et al., 1998 Science 280, 1256–1258, with modifications. All cells were derived from either explant cultures or enzyme digests of skin biopsies and were cyropreserved and thawed at least 48 hours prior to nuclear transfer. Cells were harvested using either pronase or trypsin at 70 to 90% confluence. Oocytes were either activated prior to fusion or immediately after fusion using ionomycin. The couplets were then cultured in cycloheximide and cytochalsin B for 6 hours. In 36 cases (84%), at least one healthy calf was produced from the initial trial which included transfer to 10 to 20 recipients for each cell line. For 4 of the 7 cases where the initial cell line failed to produce a live calf, a new cell line was derived and the process repeated. In one case where the data are available from the second cell line, 5 live calves were produced from 20 recipients receiving embryos (25%). Results from the other repeated cell lines are pending. For 5 of the different genotypes, nuclear transfer was done at about the same time using two different cell lines, and 4 of these have produced healthy calves from both cell lines. In one case, one cell line produced live calves, and no calves were produced from the other cell line. In total, 167 calves were born, of which 107 are alive and healthy as of this writing (64%), and range in age from 1 to 25 months. There are 86 calves older than 6 months of age and no losses have occurred as calves have aged into early adulthood. Forty-four (26%) of the calves were stillborn, failed to convert to neonatal circulation or were euthanized within 48 hours of birth. The most frequent reason for euthanasia was severe contracture of the limbs (arthrogryposis). This defect occurred even within cell lines that also gave rise to healthy calves, although it was more prevalent with certain cell lines. Other complications among the normal calves born were those of an abnormally large umbilicus or umbilical vessels. In addition, 16 calves were lost after the first 48 hours (13%). Two of these losses were due to accidents and 9 of them were due to complications from umbilical infections. The other 5 calf loses resulted from complications common to young calves such as clostridial infection and ruptured abomasum. Recent improvements in cell line derivation and embryo culture techniques, as well as a higher incidence of natural birth and improved neonatal management, have resulted in healthy calf production efficiencies (from embryos transferred) greater than 30% for 5 independent genotypes. The number of healthy calves produced per embryo transferred was 11 of 20 (55%), 5 of 10 (50%), 5 of 10 (50%), 4 of 11 (36%), and 3 of 10 (30%), for each of these genotypes, respectively. There was no correlation between the efficiency of blastocyst production and pregnancy outcome for the cell lines evaluated in this study. In conclusion, the efficiency of live healthy calf production using somatic cell nuclear transfer remains variable, depending on both the cell line and the genotype. However, efficiencies approaching those obtained using conventional embryo transfer is possible.

CHROMOSOMES AND DNA OF MICROTUS II. CONFIRMATION OF DELETION OF CONSTITUTIVE HETEROCHROMATIN IN M. AGRESTIS CELLS IN VITRO

1977 ◽  
Vol 19 (3) ◽  
pp. 537-541 ◽  
Author(s):  
J. E. K. Cooper
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Cell Lines ◽  
X Chromosome ◽  
Sex Chromosomes ◽  
Constitutive Heterochromatin ◽  
The Other ◽  
Microtus Agrestis ◽  
C Banding

The distribution of constitutive heterochromatin has been examined by C-banding in two somatic cell lines, grown in vitro, from a female Microtus agrestis. One line retains one intact X chromosome together with the short arm of the other X chromosome, while the other cell line retains only the short arm of one X chromosome. Thus, each cell line has lost substantial amounts of heterochromatin from the sex chromosomes, but this material has been deleted from the cells, and not translocated to other chromosomes. Nonetheless, both cell lines continue to propagate well in vitro.

87 VARIATION IN HEMATOLOGICAL PROFILES BETWEEN BOVINE SOMATIC CELL NUCLEAR TRANSFER CLONES AND CONTEMPORARIES FROM BIRTH TO ADULTHOOD

2009 ◽  
Vol 21 (1) ◽  
pp. 144 ◽  
Author(s):  
M. P. Green ◽  
C. Couldrey ◽  
M. C. Berg ◽  
D. N. Wells ◽  
R. S. F. Lee
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Repeated Measures ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Hematological Parameters ◽  
Cell Counts ◽  
Normal Ranges ◽  
And Control ◽  
Over Time

The hematological characterization of clones derived by somatic cell nuclear transfer (SCNT) has not been extensively reported. Studies show that, generally, hematological parameters are within normal ranges, although distinct divergence between specific cohorts of clones and contemporaries exist. The aim of this study was to identify similarities and differences between cohorts of bovine clones and control animals and analyze the variations over time as the collective cohorts mature. Hematological profiles of 47 clones derived from 4 cell lines and 23 of their age- and sex-matched contemporary controls were compared. These donor cell lines were from 2 beef (male, n = 30) and 2 dairy (1 male, n = 9 and 1 female, n = 8) breeds and derived from myogenic cells, skin fibroblasts, and granulosa cells. Matched contemporaries, analyzed as one group, were produced via natural mating (n = 5) and AI (n = 14), with an additional in vitro-produced (IVP) group (n = 4) in the female cohort. All animals were subjected to similar management, nutrition, and environmental conditions. Serial samples were collected from birth until 15 months. Samples were assessed for the standard hematological parameters and cell morphology by a commercial clinical lab. Parameters were analyzed by one-way or as repeated measures ANOVA. The mean values for erythroid, myeloid, and lymphoid parameters were within normal ranges for both SCNT and controls, indicative of normal physiology. Red blood cells (RBC) from SCNT and control calves showed anisocytosis, poikilocytosis, cell fragmentation, and stippling, with a greater prevalence found in SCNT than in the controls. These abnormal morphologies were still evident in SCNT animals at 15 months of age, suggestive of delayed or incomplete erythroid maturation. Numbers of RBC, mean corpuscular volume (MCV), and hemoglobin (MCH) were different (P < 0.0001) between the collective SCNT cohorts and control animals over time, irrespective of genetics, sex, or breed. Taken together, these data suggest that erythropoiesis is generally perturbed in SCNT animals. In beef SCNT lines, platelet numbers were consistently different (P < 0.0001) from controls. White blood cell counts (WBC) were greater (P < 0.05) collectively in SCNT, although within the normal range, and the differential WBC changed with age (P < 0.05). Lymphocyte counts were greater (P < 0.05) in the collective SCNT cohorts. Further differences were seen in myeloid counts between specific SCNT and control cohorts. The greater variance evident in the myeloid parameters of SCNT animals was presumably because of an increased incidence of transient infections or inflammation in these animals. In summary, although most parameters were within the normal ranges over time, SCNT animals commonly display altered RBC, MCV, MCH, WBC, and lymphocyte parameters, which may be linked to cloning per se. This could partially explain the greater susceptibility of SCNT animals to external stressors. Supported by FRST contract C10X0311 and NRCGD.

43 PRODUCTION OF CLONED BLASTOCYSTS USING EPITHELIAL CELLS CULTURED FROM BOVINE SEMEN

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
J. Liu ◽  
M. E. Westhusin ◽  
D. C. Kraemer
Keyword(s):  
Epithelial Cells ◽  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Bovine Semen ◽  
Cells Cultured

Somatic cells in semen could be a valuable source of nuclei for cloning animals by somatic cell nuclear transfer, especially when other ways of obtaining somatic cells are not available. The usefulness of the cells cultured from bovine semen for nuclear transfer was evaluated in the present study. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and a crossbreed rodeo bull. All of the samples were processed immediately, and somatic cells were isolated by centrifuging through 20%, 50%, and 90% percoll columns (Nel-Themaat et al. 2005 Reprod. Fertil. Dev. 17, 314–315). Somatic cell lines were obtained from 7 of the 12 ejaculates. These cell lines have classic epithelial morphology, express cytokeratin and vimentin, and proliferate well in the medium we previously designed for the epithelial cells in ovine semen (Jie Liu et al. 2007 Biol. Reprod. special issue, 177–178). Cell lines from three bulls that had been cultured in vitro for 1–2 months were used in the cloning experiments. Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory in warm saline solution within 2–4 h. Compact cumulus–oocyte complexes with evenly distributed cytoplasm were selected and matured for 18 h at 38.5�C with 5% CO2 in humidified air. Cumulus cells were removed by pipetting in 0.3% hyaluronidase solution (Sigma Chemical Co., St. Louis, MO, USA) for 5 min. Oocytes were selected for the presence of a first polar body and stained in 5 µg mL–1 Hoechst 33342 (Sigma) and 5 µg mL–1 cytochalasin B (Sigma) for 10–15 min before enucleation. Successful enucleation was confirmed by brief exposure of the oocytes to ultraviolet light. Epithelial cell lines cultured to 90–100% confluence were trypsinized, and a single cell was inserted into the perivitelline space of an oocyte. Fusion was induced by applying two 1.8–1.9 kV cm–1, 20 µs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in fusion medium comprising 0.28 m Mannitol (Sigma), 0.1 mm CaCl2 (Sigma), and 0.1 mm MgSO4 (Sigma). One and half to 2 h post fusion, activation was induced by applying two 0.3 kV cm–1, 55 µs direct-current pulses in the fusion medium, followed by incubation in 10 µg mL–1 cycloheximide (Sigma) and 5 µg mL–1 cytochalasin B for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5�C. The embryos were washed three times and cultured in commercially available G1/G2 medium (Vitrolife, Inc., Englewood, CO, USA) for up to 10 days. Blastocyst development rates using somatic cells from three of the bulls, 1-year-old Charolais, 6-year-old Brahman, and 8-year-old Brahman, were 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused one-cell embryos, respectively. Of these blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. The present study shows that epithelial cells cultured from bovine semen can be used to produce blastocyst-stage embryos by somatic cell nuclear transfer.

scholarly journals Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lucia Natalia Moro ◽  
Diego Luis Viale ◽  
Juan Ignacio Bastón ◽  
Victoria Arnold ◽  
Mariana Suvá ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Myostatin Gene ◽  
Clonal Cell ◽  
Fetal Fibroblasts ◽  
Clonal Cell Lines

Abstract The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

30 IN VITRO EXPRESSION OF BAX AND BCL2 GENES IN NUCLEAR DONOR SKIN FIBROBLAST, CUMULUS, AND GRANULOSA CULTURED CELLS DURING CULTURE AND THEIR SOMATIC CELL NUCLEAR TRANSFER-CLONED EMBRYOS IN INDIAN BUFFALOES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 115
Author(s):  
N. Gupta ◽  
A. Pandey ◽  
S. C. Gupta
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Granulosa Cells ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Skin Fibroblast ◽  
Cultured Cells ◽  
Donor Cell ◽  
Cumulus Cells

Somatic cell nuclear transfer (SCNT) involves functional changes in the genome which result in low efficiency for the production of viable and cloned embryos. It is primarily due to incomplete reprogramming of genome of donor cell nuclei in the reconstructed embryos (Vassena et al. 2007 Dev. Biol. 304, 75–89). Expression of BCL2 and Bax can be correlated with apoptosis. BCL2 inhibits apoptosis by regulating the release of cytochrome-c and other proteins from mitochondria (Keep et al. 2007 EMBO J. 26, 825–834). Antiapoptotic BCL2 is antiproliferative by facilitating G0. Bax is proapoptotic and accelerates S-phase progression. The dual functions in apoptosis and cell cycle are coordinately regulated by the BCL2 family and suggest that survival is maintained at the expense of proliferation (Zinkel et al. 2006 Cell Death Differ. 13, 1351–1359). The aim of this study was to estimate the relative expression of BCL2 oncogene and Bax gene in regulating apoptosis, in skin fibroblast, cumulus, and granulosa cells in culture, so that ideal-type donor cell lines are developed for higher success rates in SCNT-derived buffalo cloning. The cell lines up to 25th passage were from all the 3 tissue types by previous method (Gupta et al. 2007 Cell Biol. Int. 31, 1257–1264). The cells between passages 5th to 15th were selected as competent donor cells and transferred into enucleated in vitro-matured oocytes from slaughter ovaries. The couplets were activated electrically (1.5 kV cm–2, 15 μs) and chemically (ionomycin, 6-DMAP, CHX, and Cyto-B) and were cultured up to blastocyst. The cDNA were prepared from the growing cells in culture at 5, 10, and 15 passages from all cell lines and SCNT-cloned blastocysts from these cell lines at respective passages for Bax and BCL2 gene expression analysis. Relative expression of these candidate genes was quantified using real-time PCR. The data was analyzed for 1-way ANOVA and post-hoc Duncan multiple range test at P ≤ 0.05 level of significance. The cell proliferation rate in cultured cells at fifth passage was higher in all the 3 cell lines and declined in subsequent passages (range from 1.06 to 0.67). The relative abundance of Bax mRNA in granulosa cell was comparable with skin fibroblasts but significanly higher than cumulus cells at respective passages. BCL2 mRNA expression was significantly upregulated in cumulus cells as compared to granulosa cells but not with skin fibroblasts. The SCNT blastocyst production rates from granulosa were highest (24.28%) as compared to fibroblast (22.6%) and cumulus (21.4%) at passage 10. Level of Bax and BCL2 mRNA in granulosa and fibroblast SCNT blastocysts was not significantly different from IVF (control), whereas cumulus-derived blastocyst showed abnormal patterns with downregulated expression of Bax mRNA and upregulated expression of BCl2 mRNA. Identification of expressed genes in cells and cloned embryos will help to investigate the causes of developmental abnormality due to deregulation of expression of important gene associated with ART.

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