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.

Aberrant expression of E-cadherin and β-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

2012 ◽  
Vol 24 (4) ◽  
pp. 588 ◽  
Author(s):  
H. R. Kohan-Ghadr ◽  
L. C. Smith ◽  
D. R. Arnold ◽  
B. D. Murphy ◽  
R. C. Lefebvre
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Target Genes ◽  
Subcellular Localisation ◽  
Aberrant Expression ◽  
Qrt Pcr ◽  
And Control ◽  
E Cadherin

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT–PCR (qRT–PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT–PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Nuclear Donor Cell Lines Considerably Influence Cloning Efficiency and the Incidence of Large Offspring Syndrome in Bovine Somatic Cell Nuclear Transfer

2013 ◽  
Vol 48 (4) ◽  
pp. 660-664 ◽  
Author(s):  
J Liu ◽  
Y Wang ◽  
J Su ◽  
Y Luo ◽  
F Quan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cloning Efficiency

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.

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

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.

33 REPRODUCTIVE CHARACTERISTICS OF CLONED DOGS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 117
Author(s):  
S. G. Hong ◽  
J. E. Park ◽  
J. T. Kang ◽  
H. J. Oh ◽  
M. J. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Venous Blood ◽  
Ovarian Follicles ◽  
Hormonal Changes ◽  
Reproductive Characteristics ◽  
Fetal Fibroblasts ◽  
And Control ◽  
Hormonal Analysis

Several cloned dogs have been successfully produced by somatic cell nuclear transfer. However, there is no investigation of its reproductive characteristics including changes of hormone and ovarian follicles in cloned dogs. Thus, this study was to examine the onset of puberty, follicular dynamics, and reproductive hormone profiles in cloned beagle dogs. Two female cloned beagles, derived from fetal fibroblasts, were compared to 2 individual age- and weight-matched female beagles produced by natural breeding. All dogs were examined twice weekly from 6 months for the presence of swelling of the vulva and serosanguineous vaginal discharge, which were used as markers of the onset of proestrus. From the first day of proestrus, jugular venous blood samples were collected twice a day for hormonal analysis. Also, from the day when progesterone concentration was >1 ng mL–1 until 5 days after ovulation, blood sampling was carried out 3 times a day. Ultrasound examination of ovaries and uterus was performed daily from the onset of proestrous until 5th day post-ovulation. Cloned female dogs (332.5 ± 1.5 days) reached puberty later than controls (300 ± 8 days). The mean number of ovarian follicles was 4.5 ± 0.5 v. 6.5 ± 0.5 for clones and controls, respectively. The largest size of ovarian follicles in clones and controls detected by ultrasound were 0.6 ± 0.03 v. 0.69 ± 0.07 cm, respectively. It took 8 days from the initiation of vulvar bleeding to the LH peak in all beagles. Moreover, there were no differences in the profile of hormonal changes (LH, FSH, estradiol, and progesterone) in cloned and control beagles. These results demonstrate that cloned dogs have normal development of reproduction. This study was financially supported by the Korean MEST, through KOSEF (grant # M10625030005-08N250300510) and the BK21 program for Veterinary Science.

Somatic Cell Nuclear Transfer–Derived Embryonic Stem Cell Lines in Humans: Pros and Cons

2013 ◽  
Vol 15 (6) ◽  
pp. 481-483 ◽  
Author(s):  
Alena Langerova ◽  
Helena Fulka ◽  
Josef Fulka
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Pros And Cons ◽  
Stem Cell Lines
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