Cellular reprogramming of adult goat fibroblast: Toward pluripotency

2016 ◽  
Author(s):  
Dharmendra Kumar ◽  
Rakesh Ranjan ◽  
Ajit P. Singh ◽  
Bikash C Sarkhel
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Cellular Reprogramming ◽  
Serum Starvation ◽  
Methyl Transferase ◽  
Donor Cells ◽  
Key Factor ◽  
Gene Transcripts

Cellular reprogramming erases the epigenetic constraints of somatic cells genome and thus considered as key factor for success of somatic cell nuclear transfer technology. To achieve the reprogramming, different strategies are used which are mostly based on arresting the cell cycle at G0 or G1 stage. The present study was based on molecular investigation of reprogrammed cells for expression of pluripotent genes that are crucial for development of cloned embryos. The fibroblast cell lines were treated by four methods to induce cellular reprogramming viz., serum starvation, Roscovitin, aphidicolin and overconfluent. These treated cell lines were used for quantification of pluripotent gene transcripts by using real time PCR machine. The results showed that the relative expression of different pluripotent genes as Oct-4 and Nanog along with DNA methyl transferase gene (Dnmt-1) was observed in four treated cells. In case of normal cells, only Dnmt-1 gene was expressed, but pluripotent genes were not expressed at detection level. The expression of pluripotent genes in the donor cells prior to nuclear transfer have significant impact on cloning as because it facilitates the expression of that gene in the resulting embryo after nuclear transfer. The finding of this study may be extended for stem cell generation as it showed that pluripotent genes could be induced in the somatic cells without any transgenic incorporation.

scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY

2006 ◽  
Vol 18 (2) ◽  
pp. 148
Author(s):  
J. F. Yang ◽  
S. H. Yang ◽  
Y. Y. Niu ◽  
Q. Zhou ◽  
W. Z. Ji
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Methylation Pattern ◽  
Serum Starvation ◽  
Global Dna Methylation ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Morula Stage ◽  
Asymmetric Methylation

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.

28 GENERATION OF REACTIVE OXYGEN SPECIES IN BOVINE CULTURED SOMATIC CELLS AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DURING MICROMANIPULATION PROCEDURES AND EARLY IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 120 ◽  
Author(s):  
H. K. Bae ◽  
J. Y. Kim ◽  
I. S. Hwang ◽  
C. K. Park ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Serum Starvation ◽  
Ros Generation ◽  
Oh Radical ◽  
Oxygen Species ◽  
Donor Cells

The present study was conducted to examine the reactive oxygen species (ROS) generation levels in the donor cells, recipient oocytes, and somatic cell nuclear transfer (SCNT) embryos during nuclear transfer procedures. Bovine ear skin cells were classified by serum starvation, confluence, and cycling cells. Bovine metaphase II (MII) oocytes matured in vitro for 22 h and denuded by vortexing were enucleated and electrofused with serum-starved donor cells, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture for 4 h. In vitro fertilization (IVF) was performed for controls. SCNT and IVF embryos were cultured in CR1aa supplemented with 3 mg mL–1 BSA for ∼36 h. Donor cells, recipient oocytes, and SCNT embryos were stained in 10 μM dichlorohydrofluorescein diacetate (DCHFDA) or 10 μM HPF dye each for 30 min at 39°C to measure the H2O2 or ·OH radical levels after various micromanipulation steps. SCNT and IVF embryos were also stained at the 1-, 2-, and 4-cell stages after 8, 24, and 42 h of fusion or insemination, respectively. The fluorescent emissions from the samples were recorded as JPEG file using a digital camera (F5.0, 4 s) attached to a fluorescent microscope with filters at 450 to 480 nm for excitation and at 515 nm for emission. The images were analysed using ImageJ software 1.37 (NIH) by the intensity of fluorescence (pixels) in each cell (total 70 to 75 cells in each group), oocyte and embryo (total 50 to 60 eggs or embryos in each group). 4 to 7 replicates were performed for each experiment, and data were analysed by Duncan′s multiple-range tests. H2O2 and ·OH radical levels of cultured somatic cells were high in confluence group and significantly low in serum starvation group (P < 0.05). During micromanipulation, H2O2 levels in recipient oocytes and SCNT embryos were increased by enucleation (37.2 pixels), electrofusion (49.7 pixels), and activation (40.6 pixels) treatments (P < 0.05) compared to that in MII oocytes (33.1 pixels), and the level of H2O2 was extremely increased immediately after electrofusion. ·OH radical levels were significantly higher during manipulation procedures (51.6 to 55.7 pixels; P < 0.05) compared to MII oocytes. During in vitro culture, the H2O2 and ·OH radical levels of SCNT embryos were significantly higher (P < 0.05) compared to IVF embryos at 1- (32.4 v. 17.3 and 52.0 v. 29.6 pixels, respectively), 2- (27.2 v. 22.0 and 33.4 v. 26.0 pixels, respectively), and 4-cell (25.1 v. 16.5 and 26.9 v. 20.7 pixels, respectively) stages. These results suggest that the culture type of donor cells can affect the ROS generation level and the cellular stress during micromanipulation procedures also can generate the ROS in bovine SCNT embryos, which may lead the cellular damages in bovine SCNT embryos. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008–313-F00067).

73 IMPROVEMENT OF CANINE CLONING EFFICIENCY BY OPTIMIZED DONOR CELL PREPARATION

2010 ◽  
Vol 22 (1) ◽  
pp. 195
Author(s):  
S. W. Park ◽  
Y. W. Jeong ◽  
J. J. Kim ◽  
K. H. Ko ◽  
S. H. Jeong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Electrical Stimulation ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Contact Inhibition ◽  
Abnormal Development ◽  
Serum Starvation ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Cloned Dog

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al. (2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1 for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author.

scholarly journals 231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

2004 ◽  
Vol 16 (2) ◽  
pp. 236 ◽  
Author(s):  
Z. Beyhan ◽  
N.L. First
Keyword(s):  
Gene Expression ◽  
Statistical Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Live Birth ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Gene Expression Patterns ◽  
Donor Cells

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.

47 SUCCESSFULLY PRODUCING CLONED MICE FROM SOMATIC CELLS OF AGED MICE VIA ESTABLISHED ntES CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 104
Author(s):  
E. Mizutani ◽  
T. Ono ◽  
L. Chong ◽  
T. Wakayama
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Chimeric Mice ◽  
Cell Nuclei ◽  
Female Mice ◽  
Aged Mice ◽  
Tail Tip ◽  
Tip Cells

Recent nuclear transfer techniques have enabled us to produce cloned animals from somatic cell nuclei in a variety of animal species and are to date the only way to obtain offspring from infertile animals. Despite very aged mice often showing an infertile phenotype, the decreasing rate of cloning success with increased age makes it almost impossible to produce cloned mice or offspring from these animals. Other studies, however, have demonstrated that ES cell lines have been established from cloned blastocysts through somatic cell nuclear transfer (ntES cells), irrespective of sex, strains, or organs. These cells are subsequently capable of differentiating into all three germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. Thus, ntES cells have received considerable attention recently in regenerative medicine. Importantly, the success rate of establishing ntES cell lines from cloned blastocysts is ten times higher than that of producing cloned mice, which may allow us to establish ntES cell lines even from such 'unclonable' aged mice. ntES cells also have the potential to be a good donor source for nuclear transfer as they have the same DNA as their donor somatic cells and can indefinitely proliferate in their undifferentiated states. In this study, we attempted to establish ntES cell lines from aged mice and analyze their normality. We then tried to produce cloned mice via nuclear transfer using established ntES cell lines. We obtained donor cells from tail-tip fibroblast cells of BDF1 and BCF1 male and female mice that were over two years old. Following nuclear transfer, we transferred a proportion of the cleaved cloned embryos to pseudopregnant ICR female mice. The remaining embryos were cultured for 72 h, and cloned embryos that developed into morulae or blastocysts were plated on feeder cells. We then examined all established ntES cell lines for normality by Oct4 and Nanog expression using immunofluorescence staining and pluripotency by chimeric mice formation, for which ntES cells were injected into fertilized ICR embryos. Finally, we attempted to produce cloned mice from the nuclei of these ntES cell lines. In each experiment, 25, 37, 73, 63, and 75 cloned embryos from aged mice tail-tip cells were used in attempts to produce cloned mice, and 20, 20, 27, 35, and 40 cloned embryos were used to derive ntES cell lines, respectively. No cloned mice were obtained by direct nuclear transfer of the aged mice tail-tip cells; however, we were successful in establishing ntES cell lines from all experiments, with an establishment rate between 10 and 25%. All established ntES cell lines expressed Oct4 and Nanog and contributed to somatic cells in chimeric mice. Some chimeric mice produced offspring derived from ntES cells after mating. We were also able to produce cloned mice even from a 2-year-old and a 9-month-old BCF1 male mouse by nuclear transfer using ntES cells as donor nuclei. These results clearly show that normal ntES cell lines can be established from infertile, aged mice and this technique can now be used to produce offspring, irrespective of donor conditions.

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.

58 PRODUCTION OF CLONED BOVINE EMBRYOS DERIVED FROM AMNIOTIC CELLS OF PREGNANT COWS

2008 ◽  
Vol 20 (1) ◽  
pp. 110
Author(s):  
S. Taniguchi ◽  
N. Hayashi ◽  
Y. Abe ◽  
D. Iwamoto ◽  
S. Kishigami ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Lamp Method ◽  
Developmental Capacity ◽  
Grand Island ◽  
Cloned Bovine ◽  
Amniotic Cells

Progeny tests are widely used for selection of sires for beef and dairy cattle. A less costly method might be to clone the sire candidates at their earliest developmental stage possible. To produce cloned bulls, we obtained amniotic cells as donors for nuclear transfer by transvaginal aspiration of pregnant cows. However, the collected cells may include some maternal cells. In this study, we examined collection methods to obtain only fetal cells from the collected fluid. We also examined the developmental capacity of the embryos cloned from these cells. Amniotic fluids were aspirated from pregnant cows by ultrasound-guided aspiration. We collected amniotic fluids from 27 pregnant Japanese black beef cattle (between 58 and 132 days of gestation). In Method 1, cells were recovered from the whole amniotic fluid (approximately 15 mL). In Method 2, the initial 5 mL of aspirated fluid was discarded and then the next 10 mL sample was collected. Cells were recovered from the collected fluids. The cells in the fluids were washed twice by centrifugation and then cultured in AmnioMAX™-II medium (GIBCO, Grand Island, NY, USA). After 3–4 passages, the sex of the cell lines was determined by the loop-mediated isothermal amplification (LAMP) method (Eiken Chemical Co., Ltd., Tokyo, Japan). For the cell lines that were determined as 'male' by the LAMP method we further analyzed the sex of individual cells (137–620 cells of each cell line) by fluorescent in situ hybridization (FISH) using a bovineY chromosome-specific probe (Kobayashi et al. 1998 Mol. Reprod. Dev. 51, 390–394). The percentage of male cells obtained from Methods 1 and 2 were 0–0.4% (from 4 animals) and 93.7–99.5% (from 6 animals), respectively. Then, we used confluent amniotic cells from 3 cell lines obtained by Method 2 as donor cells for nuclear transfer and examined the developmental capacity of the cloned embryos. Bovine fibroblasts cultured under serum starvation were used as a control. The cells were electrically fused (2.7 kV cm–1, 11 µs, 2 times) with enucleated bovine oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF until 168 h post-activation. The data were analyzed with Fisher's protected least-squares difference (PLSD) test following ANOVA. The rates of fusion, cleavage, and development to the blastocyst stage of the cloned embryos were the same as those of the control embryos (78% v. 81%, 75% v. 75%, and 22% v. 27%, respectively; P > 0.05). Furthermore, the rate of male blastocysts derived from the cloned embryos with the three cell lines was 95% (19/20). These results indicate that the amniotic fluids collected from pregnant cows by Method 2 contained fewer maternal cells, and that the embryos cloned from the cells developed in a manner similar to that of embryos cloned from the fibroblasts. This work was supported byWakayama Prefecture CREATE, JST.

57 NUCLEAR TRANSFER WITH RABBIT EMBRYONIC STEM CELLS: SERUM-STARVATION IMPROVES THE QUALITY OF CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
V. Zakhartchenko ◽  
F. Flisikovska ◽  
R. Hao ◽  
S. Li ◽  
A. Kind ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Serum Starvation ◽  
Chromosomal Dna ◽  
Culture Dish

Rabbit cloning by NT with somatic cells is so far a rather inefficient process. However, this technology is urgently required to generate rabbits with a humanized immune system as a source of human polyclonal antibodies. Embryonic stem cells (ESCs) have a number of advantages over somatic cells as tools for cell-mediated transgenesis including long periods of proliferation in vitro, higher frequency of homologous recombination between exogenous and chromosomal DNA, and less requirements for reprogramming (Rideout et al. 2000 Nat. Genet. 24, 109–110). To improve rabbit cloning we have derived and characterized 19 putative rabbit ESC lines and tested cells from 6 lines as donors for NT. First, we assessed in vitro development of NT embryos. Blastocyst rates varied in the range of 6–68% depending on the particular cell line and passage number, but the quality of the resultant embryos was worse compared to NT embryos derived from adult fibroblasts [hatched blastocysts: 13/214 (6%) v. 36/86 (42%), respectively]. Transfer of NT embryos derived from the ESC line showing the highest development to blastocysts into recipients resulted only in implantations (70%, 7/10) but not in offspring. Assuming that poor quality of NT embryos derived from ESCs could be due to the incompatibility between cell cycles of donor and recipient cells we used serum starvation to make ESCs more suitable for nuclear transfer. Serum starvation of one of the ESC lines (0.5% FCS for 3 days) greatly improved the quality of cloned embryos compared to those derived from non-starved cells of the same ESC line as indicated by the high proportions of hatched [38/151 (25%) v. 4/153 (3%)] and attached [25/151 (17%) v. 0%] to the surface of a culture dish blastocysts. Moreover, some of these blastocysts grew in vitro for 14–25 days. Our study provides evidence that the quality of NT embryos derived from ESCs can be significantly improved using serum starvation of donor cells suggesting possible effect of this treatment on the cell cycle synchronization. We are currently testing whether serum starvation of ESCs would also improve post-implantation development of rabbit NT embryos. This work is supported by Roche Diagnostic GmbH.

Sign in / Sign up

Export Citation Format

Share Document