64 PRODUCTION OF TRANSGENIC CLONED PIGS BY MEANS OF SOMATIC CELL NUCLEAR TRANSFER USING KUSABIRA-ORANGE GENE-TRANSFECTED CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 150
Author(s):  
H. Matsunari ◽  
M. Kurome ◽  
R. Tomii ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Control Cell ◽  
Retroviral Vector ◽  
Transfected Cells ◽  
Cell Numbers ◽  
Red Fluorescence ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Significant Difference

Cloned pigs that express cell markers such as fluorescent proteins (Vintersten et al. 2004 Genesis 40, 241–246) are useful in biomedical research in areas such as cell/tissue transplantation and regenerative medicine. In this study, we attempted to produce transgenic cloned pigs from porcine fetal fibroblasts which carry the gene of red fluorescent protein, humanized Kusabira-Orange (huKO). We examined whether huKO-transfected cells are suitable as nuclear donors for somatic cell cloning, and whether red fluorescence can be detected in the cloned embryos. We used porcine fetal fibroblasts transfected with the huKO gene and a retroviral vector as the nuclear donor cells. Non-transfected cells were used as the control. Cumulus–oocyte complexes collected from slaughterhouse ovaries were in vitro-matured in NCSU23 medium to produce recipient oocytes. Nuclear transfer was conducted using a previously reported method (Kurome et al. 2003 Cloning Stem Cells 5, 367–377); the following parameters which determine the overall efficiency of nuclear transfer were investigated: (1) fusion rate between the donor cells and recipient oocytes, (2) rates of normal cleavage and blastocyst formation of the NT embryos, and (3) cell numbers in each blastocyst. A DC pulse (190 V mm-1) was used for electric fusion, and NCSU23 or PZM-5 medium was used for culturing the cloned embryos. The NT embryos on Day 7 were examined under a fluorescence microscope (G excitation) in order to evaluate the expression of red fluorescence. Some cloned embryos at the 1- to 8-cell stage (Day 1 or 2) were transferred into oviducts of estrus-synchronized recipient gilts. There was no significant difference (chi-square test) between the huKO and the control groups in the rate of fusion (132/151, 87.4% vs. 134/147, 91.2%, respectively) and cleavage rate (78/132, 59.1% vs. 86/134, 64.2%, respectively). A significantly greater percentage of huKO cell-derived embryos developed into blastocysts than did control cell-derived embryos (37/132, 28.0% vs. 20/134, 14.9%, respectively; P < 0.05). However, there was no significant difference in the blastocyst cell numbers (Student's t-test: 48.6 ± 4.8 vs. 42.3 ± 4.9, respectively). Of the 132 NT embryos, 116 (87.9%) expressed red fluorescence. The percentage of blastocysts expressing red fluorescence was 94.6% (35/37). These results demonstrate that it is possible to obtain cloned blastocysts at a high rate by nuclear transfer of cells that have been transfected with huKO using a retroviral vector, and that it is possible to observe the expression of red fluorescence in cloned embryos. With respect to the cloned embryos that did not show expression of red fluorescence, we hypothesize that this was the result of a small proportion (<1%) of donor cells which also lacked red fluorescence expression. An ultrasonic echo examination has confirmed that all 3 of the recipients which had received 93 to 119 embryos became pregnant. This study was supported by PROBRAIN.

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P < 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P < 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

36 CLONED BEAGLE DOGS DERIVED FROM FETAL FIBROBLASTS BY NUCLEAR TRANSFER

2008 ◽  
Vol 20 (1) ◽  
pp. 98 ◽  
Author(s):  
S. G. Hong ◽  
G. Jang ◽  
M. K. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Coat Color ◽  
Cell Types ◽  
Beagle Dogs ◽  
Black And White ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Identical Nucleus

Somatic cell nuclear transfer (SCNT) has been successfully performed in various mammals including sheep, cow, pig, and mouse using a variety of somatic cell types as nuclear donors. Several reports of livestock SCNT indicate that fetal fibroblasts are superior to adult fibroblasts as donor cells. In canine SCNT, however, only adult ear fibroblasts have been used as donor cells (Lee et al. 2005 Nature 436, 641; Jang et al. 2007 Theriogenology 67, 941–947). Accordingly, in the present study, we evaluated the ability of canine fetal fibroblasts to support fetal development to term after nuclear transfer. For SCNT, in vivo-matured oocytes flushed (approximately 72 h after ovulation) from the oviducts of six estrus females were used. Donor cells (fetal fibroblasts) were isolated from the fetus of a beagle bitch obtained at 28 days after artificial insemination. Before using fetal fibroblasts as donor cells, sex was determined by SRY gene detection using PCR. Oocytes were enucleated, microinjected with a female fetal fibroblast, fused by electrical stimulation, and activated chemically (Jang et al. 2007). A total of 50 cloned presumptive embryos were transferred (Day 0) into the oviducts of two naturally synchronous recipient bitches. One pregnancy, detected by ultrasonography on Day 23, was maintained to term and two healthy female puppies weighing 250 and 260 g were born by natural delivery on Day 60. They were genotypically identical to the donor cells, and had phenotypically similar black and white coat color patterns. Analysis of their mtDNA distribution showed that mtDNA in the two cloned beagles originated from one of the six oocyte donor dogs. In conclusion, our results demonstrate the potential of using fetal fibroblasts to facilitate nuclear transfer in the dog. The cloned beagle dogs, which had identical nucleus and mitochondrial DNA, will be provided for biomedical research as bioresources. This study was financially supported by KOSEF (grant # M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

2012 ◽  
Vol 24 (1) ◽  
pp. 128
Author(s):  
G. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mononuclear Cells ◽  
Donor Cell ◽  
Foreign Gene ◽  
Beagle Dogs ◽  
Fetal Fibroblasts ◽  
Significant Difference

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
Y. H. Choi ◽  
Y. G. Chung ◽  
D. D. Varner ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Injection ◽  
Donor Cell ◽  
Mixed Gas ◽  
Blastocyst Development ◽  
Donor Cells ◽  
Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

74 STUDY OF TRANSGENIC GOATS EXPRESSING HUMAN GRANULOCYTE - MACROPHAGE COLONY-STIMULATING FACTOR IN THE MAMMARY GLAND

2007 ◽  
Vol 19 (1) ◽  
pp. 154
Author(s):  
H. S. Park ◽  
S. Y. Jung ◽  
S. H. Park ◽  
J. K. Park ◽  
J. S. Lee ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Colony Stimulating Factor ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Macrophage Colony Stimulating Factor ◽  
Transgenic Goats ◽  
Macrophage Colony ◽  
Stimulating Factor

Somatic cell nuclear transfer (SCNT) is one of the most useful methods for production of transgenic animals. While both adult and fetal fibroblasts have been used in SCNT, adult cells provide the opportunity to use donor cells from an animal with a proven high yield of milk production. In this study, in vitro development and pregnancy rates were compared following the use of transfected fibroblasts that were obtained from adult and fetal tissues. Ear and fetal fibroblasts were collected from Saanen goats and cultured in serum-starvation condition (TCM-199 + 0.5% FBS) until cell confluence. Linearized DNA containing the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene that is targeted for expression in the mammary gland by the β-casein promoter (pBC1/hGM-CSF) was transfected into the cells using lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA, USA). Transfected colonies were selected with G418 for 14 days. Well-separated colonies were isolated and screened for the presence of transgene by PCR and southern blotting. Recipient oocytes were surgically collected by flushing the oviducts of FSH-stimulated goats at 35 h after hCG injection. The zonae pellucidae of the oocytes were partially drilled using a laser system and each somatic cell was individually transferred into the enucleated oocyte. The couplets were electrically fused and activated by ionomycin + 6-DMAP. The reconstructed embryos were cultured in mSOF medium containing 0.8% BSA at 39�C in an atmosphere of 5% CO2, 5% O2, 90% N2 for 12 to 15 h. Nuclear transfer embryos (2- to 4-cell stages) were surgically transferred into the oviducts of recipients that were in either natural or induced estrus. Pregnancy was diagnosed by progesterone assay and ultrasound on Days 21, 42, and 60 of pregnancy. The type of donor cells, ear or fetal fibroblasts, affected neither the fusion rate (54/65, 83.1% vs. 89/116, 76.7%, respectively) nor the cleavage rate (19/54, 35.2% vs. 37/89, 41.6%, respectively). A total of 55 embryos derived from ear fibroblasts and 84 embryos derived from fetal fibroblasts were transferred into 9 and 14 recipients, respectively. No pregnancy was observed in recipients that received NT embryos derived from ear fibroblasts (0/9); however, 5 (2 in natural estrus and 3 in induced estrus; 35.7%), 3 (1 in natural and 2 in induced estrus; 21.4%), and 2 (2 in induced estrus; 14.3%) of 14 recipients that received NT embryos derived from fetal fibroblasts were confirmed pregnant on Days 21, 42, and 60, respectively. These results imply that the type of donor cells for nuclear transfer may be directly correlated with the success rate. More studies will be needed to examine factors affecting production of transgenic goats and to improve results obtained using adult donor cells.

66EFFECTS OF CONTACT INHIBITION INTERVALS V. SERUM DEPRIVATION ON DEVELOPMENT OF PORCINE NUCLEAR TRANSFER DERIVED EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 155
Author(s):  
B. Petersen ◽  
M. Hoelker ◽  
W. Kues ◽  
H. Niemann
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Serum Deprivation ◽  
T Test ◽  
Contact Inhibition ◽  
The Other ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Significant Difference ◽  
Blastocyst Rate

Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco‘s Modified Eagles Medium (DMEM) supplemented with 2mM glutamine, 1% non-essential amino acids, 0.1mM mercaptoethanol, 100UmL−1 penicillin, 100mgmL−1 streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24h, 48h and 72h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456–461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24h, 48h and 72h, respectively). After nuclear transfer (for each CI group n=336–384 reconstr. embryos; SD n=215) there was a statistically significant difference in the fusion rate between 48h CI and SD cells (74.8% v. 87.5%, t-test P&lt;0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24h CI and 48h CI (17.4%; 9.1%, 9.6%, t-test P&lt;0.050), there was no difference between SD and 72h CI and within the CI groups (72h CI 10.6%). Four transfers of reconstructed embryos (72h CI, n=138–163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n=96–110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).

43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 130
Author(s):  
H.-F. Jin ◽  
B. Mohana Kumar ◽  
J.-G. Kim ◽  
H.-J. Song ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% � 4.6 vs. 52.2% � 5.4 and 50.8% � 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% � 2.5 and 18.5% � 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% � 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 � 5.2 and 0.38 � 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 � 5.5 and 0.18 � 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 � 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 � 1.8) and in IVF embryos (4.6 � 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

55 CONSTRUCTION AND IN VITRO DEVELOPMENT OF FOLLISTATIN TRANSGENIC PORCINE EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 133
Author(s):  
Y. S. Mu ◽  
D. D. Jiang ◽  
Y. J. Huan ◽  
J. Zhu ◽  
H. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Cell ◽  
Pcr Analysis ◽  
Cleavage Rate ◽  
Cmv Promoter ◽  
Transfected Cells ◽  
Blastocyst Rate

Data from mice showed that follistatin can block myostatin activity in vivo and follistatin overexpressed in transgenic mice can induce dramatic increases in muscle mass. In order to determine whether this effect of follistatin have the same results in swine, we design experiment to make a follistatin transgenic porcine by somatic cell nuclear transfer. We hope to get a follistatin transgenic porcine that has double muscle mass. The present study aimed to construct follistatin transgenic embryos by somatic cell nuclear transfer. A follistatin (FST) expression vector, pFST-1, which contains a porcine FST cDNA (with modification of codon usage) driven by the human cytomegalovirus (CMV) promoter, was constructed. A SV40-neo expression cassette as a selection marker was inserted into pFST-1 to generate pST101, which was transfected into early-passage male primary porcine fetal fibroblast cells by the lipofection method; the transfected cells were selected with 400 mg mL–1 G418. The G418-resistant colonies were pooled. Cell genome DNA was extracted and PCR analysis showed that CMV promoter, follistatin CDS, and SV40 polyA can be detected, indicating that the expression construct had integrated in cell genome. RT-PCR analysis showed that pST101 cells contained higher amounts of FST cDNA compared with the non-transfected cells, indicating that the follistatin was overexpressed in the primary porcine cells. Then, pST101 cell as donor cell and non-transfected cells as control donor cell were used to construct somatic cell nuclear transfer (SCNT) embryos. All of these SCNT embryos were cultured in Porcine Zygote Medium-3. The cleavage rate and blastocyst rate was assessed on 48 h and Day 6. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). The cleavage rate of pST101 cell and control cell derived SCNT embryos were 83.6 ± 2.0% (267/318) and 84.0 ± 2.2% (139/167) respectively, there were no significant (P > 0.05) differences between them. The blastocyst rate from pST101 cell and control cell derived SCNT embryos were 21.4 ± 1.8% (65/318) and 15.0 ± 3.0% (26/167) respectively, the SCNT embryos derived from pST101 cell showed a higher (P < 0.05) blastocyst rate. These results indicated that follistatin transgenic porcine embryo can be successfully constructed and can develop in vitro to blastocyst with a higher rate than control SCNT embryo. Further study will be focused on the full development in vivo of the transgenic embryos and phenotype of transgenic piglets. This work was supported in part by the National High-tech Research and Development Program of China (2008AA101006) and Major Projects for Transgenic Animals of China (2008ZX08006-002).

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽  
2011 ◽  
Vol 20 (1) ◽  
pp. 67-72 ◽  
Author(s):  
So Gun Hong ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
Min Jung Kim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Transfected Cells ◽  
Fetal Fibroblasts ◽  
Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

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