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.

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.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
V. Zakhartchenko ◽  
F. Yang ◽  
R. Hao ◽  
E. Wolf
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Epigenetic Mark ◽  
Cloning Efficiency ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Fetal Fibroblasts

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P < 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P < 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P < 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P < 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, 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.

259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
B. Mohana Kumar ◽  
H. F. Jin ◽  
J. G. Kim ◽  
S. A. Ock ◽  
H. J. Song ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Dna Hypomethylation ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Donor Cells ◽  
Fetal Fibroblasts

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 �m, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 �m 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P &lt; 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 � 5.2% vs. 65.8 � 5.3% and 69.3 � 4.4%, respectively). Similarly, significantly (P &lt; 0.05) higher blastocyst rates were observed in IVF embryos (27.2 � 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P &lt; 0.05) higher rates of blastocysts (21.3 � 2.2%) than NT embryos from PFF (14.8 � 1.9%). NT embryos from PFF + 5-azaC (33.8 � 4.1) had significantly (P &lt; 0.05) higher total cell numbers than from PFF (24.6 � 3.5), but did not differ in the proportion of apoptotic cells (6.9 � 1.8% and 7.2 � 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 � 3.8, 2.7 � 0.8%, and 53.9 � 3.5, 1.2 � 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

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 &lt; 0.05). However, there was no significant difference in the blastocyst cell numbers (Student&apos;s t-test: 48.6 &plusmn; 4.8 vs. 42.3 &plusmn; 4.9, respectively). Of the 132 NT embryos, 116 (87.9&percnt;) expressed red fluorescence. The percentage of blastocysts expressing red fluorescence was 94.6&percnt; (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 (&lt;1&percnt;) 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.

Somatic cell nuclear transfer in pigs: recent achievements and future possibilities

2007 ◽  
Vol 19 (2) ◽  
pp. 403 ◽  
Author(s):  
Gábor Vajta ◽  
Yunhai Zhang ◽  
Zoltán Macháty
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Direct Evidence ◽  
Research Area ◽  
Genetically Engineered ◽  
Transgenic Pigs ◽  
Actual Performance ◽  
Donor Cells

During the past 6 years, considerable advancement has been achieved in experimental embryology of pigs. This process was mainly generated by the rapidly increasing need for transgenic pigs for biomedical research purposes, both for future xenotransplantation to replace damaged human organs or tissues, and for creating authentic animal models for human diseases to study aetiology, pathogenesis and possible therapy. Theoretically, among various possibilities, an established somatic cell nuclear transfer system with genetically engineered donor cells seems to be an efficient and reliable approach to achieve this goal. However, as the result of unfortunate coincidence of known and unknown factors, porcine embryology had been a handicapped branch of reproductive research in domestic animals and a very intensive and focused research was required to eliminate or minimise this handicap. This review summarises recent achievements both in the background technologies (maturation, activation, embryo culture) and the actual performance of the nuclear replacement. Recent simplified methods for in vivo development after embryo transfer are also discussed. Finally, several fields of potential application for human medical purposes are discussed. The authors conclude that although in this early phase of research no direct evidence can be provided about the practical use of transgenic pigs produced by somatic cell nuclear transfer as organ donors or disease models, the future chances even in medium term are good, and at least proportional with the efforts and sums that are invested into this research area worldwide.

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.

scholarly journals 75 RABBIT NUCLEAR TRANSFER WITH CULTURED SOMATIC CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 187 ◽  
Author(s):  
F. Yang ◽  
B. Kessler ◽  
S. Ewerling ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Nuclear Transfer ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Electric Pulses ◽  
Donor Cells ◽  
Fetal Fibroblasts

Cloned rabbits have been obtained by somatic cell nuclear transfer (SCNT) only with fresh, non-cultured cumulus cells (Chesne et al. 2002 Nat. Biotechnol. 20, 366–369). For the purpose of generating transgenic animals by SCNT, donor cells must be cultured and modified prior to use as nuclear donors. The objective of this study was to optimize the SCNT procedure using cultured cumulus or fibroblast cells. MII oocytes were harvested from superovulated Zika rabbits, and maternal chromosomes were removed by demecolcine-assisted enucleation (Yin et al. 2002 Biol. Reprod. 67, 442–446). Two types of somatic cells originating from Ali/Bass rabbits were used as nuclear donors: cumulus cells collected from in vivo-matured oocytes and cultured for 1–5 passages, and primary fetal fibroblasts obtained from Day 16 fetuses and grown to confluence or starved for 4–5 days. Somatic donor cells and recipient cytoplasts were fused with 2 electric pulses (1.95 kV/cm, 25 μs each, 1 s interval). Twenty to 40 min after fusion, cloned embryos were activated first with the same electropulses as for fusion, and then immediately followed by 1 h incubation in 2 mM 6-dimethylaminopurine and 5 μg/mL cytochalasin B in culture medium (B2 medium supplemented with 10% FCS). Cloned embryos were either transferred at the 2- and 4-cell stage to asynchronized recipients or cultured in vitro for 6 days. Data were compared using chi-square test, and differences were considered significant when P < 0.05. Our results demonstrate that cloned rabbits can be produced by SCNT with cultured cells but the efficiency of this technique is still very low irrespective of the type of donor cells. Table 1. Development of cloned embryos derived from somatic cells This research was supported by the Therapeutic Human Polyclonals, Inc.

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.

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