scholarly journals Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Yu-Guo Yuan ◽  
Liyou An ◽  
Baoli Yu ◽  
Shaozheng Song ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Goat Milk ◽  
Cell Stage ◽  
Specific Expression ◽  
Transgenic Cell ◽  
Donor Cells ◽  
Transgenic Goats

To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goatβ-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%;P<0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to expressin vitroandin vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

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.

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 &lt; 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 &lt; 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 &lt; 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 &lt; 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.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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 &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 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.

394 CHARACTERIZATION OF BOVINE ADULT-DERIVED ADIPOSE STEM CELLS FOR USE IN NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 353
Author(s):  
A. A. Picou ◽  
J. Wilson ◽  
B. Dresser ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Skin Fibroblasts ◽  
Chi Square ◽  
Adult Cattle ◽  
Donor Cells ◽  
Significant Difference ◽  
Chi Square Analysis

Adipose tissue is an abundant source of adult-derived cells that have displayed multipotent properties in vitro. The goal of this research was to study the characteristics of bovine adipose-derived adult stem (ADAS) cells to determine the feasibility for use in NT. Adipose tissue was isolated from the brisket of adult cattle postmortem. Cells were isolated by incubation for 2 h with 0.25% collagenase solution, separation of stromal cells by centrifugation, and selection by adherence to plastic. The lifespan and growth characteristics for culture conditions were determined by a 2 × 2 factorial with DMEM or DMEM:F12 and with or without growth factor (GF) supplementation.A two-way ANOVA, followed by multiple pair-wise comparisons using Tukey’s test when applicable, was used to detect differences in population doublings (PD) until senescence for media treatments and GF supplementation. Dulbecco’s modified Eagle’s medium with GF supported significantly less (PD) (P > 0.05) than DMEM : F12. The average lifespan was approximately 30 PD, with a cell length of 48 h until passage 8 (P8). As cells approached replicative senescence, the cell cycle length was inconsistent. Two ADAS and one adult-derived skin fibroblast cell lines from different animals were subjected to differentiation conditions for adipocytes, chondrocytes, and osteoblasts at P2, P6, and P11. Differentiation was confirmed by histological staining. Passage 2 ADAS cells differentiated more efficiently than did P6, P11, or skin fibroblasts. Global levels of DNA methylation and histone acetylation were analyzed from P1 to P6 in 3 sets of cell lines consisting of ADAS and skin cells from the same animals by immune staining and flow cytometry. There was no significant difference (P > 0.05) between cell types by one-way ANOVA. Nuclear transfer was performed using ADAS cells as donor cells and commercially supplied oocytes. Mature, enucleated oocytes were reconstructed with either adult skin fibroblasts or ADAS cells. The percentage of cleaved and blastocysts from ADAS cells (62% and 8%, n = 163) and skin fibroblasts cells (42% and 8%, n = 170) were not different (P > 0.05) by chi-square analysis. Interspecies NT was attempted with eland (Taurotragus oryx) ADAS cells and enucleated bovine oocytes. Two groups of enucleated oocytes were reconstructed with bovine (n = 234) and eland (n = 290) ADAS cells. There was no significant difference between the number of cleaved embryos (38% and 39%) or blastocysts formed by chi-square analysis. A total of 3 interspecies embryos (1%) and 5 bovine embryos (14%) developed to blastocysts. Bovine ADAS cells are not more efficient than bovine adult-derived skin fibroblasts as donor cells, but they do represent a viable option for use in NT because of their higher in vitro development. Eland ADAS cells resulted in development to the blastocyst stage after interspecies NT.

101 DEVELOPMENTAL ARREST AND CYTOSKELETAL ANOMALIES OF RAT EMBRYOS RECONSTRUCTED BY SOMATIC CELL NUCLEAR TRANSFER

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
J. G. Yoo ◽  
S.-P. Demers ◽  
L. Lian ◽  
L. C. Smith
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Alpha Tubulin ◽  
Rat Embryos ◽  
Developmental Arrest ◽  
Donor Cells ◽  
G2 Phase

Many factors influence success rates in animal cloning by somatic cell nuclear transfer (SCNT), including cell cycle stage of the donor cells and recipient oocyte, the procedure of micromanipulation, and the activation protocol. It has been reported that the development of rat embryos is inhibited at the 2-cell stage during in vitro culture and that microtubule (MT) distribution is involved in the developmental arrest. This study was conducted to determine the effects of oocyte activation methods and the cell cycle coordination for cloning rats from fetal fibroblasts (FF). Recipient oocytes were activated with cycloheximide, 6-dimethylaminopurine, and roscovitine and used either before (metaphase-II, MII) or after (telophase-II, TII) activation for fusion with FF at the G0/G1-phase, metaphase, and S/G2-phase. Moreover, enucleated zygotic and parthenogenetic ooplasts were used for serial cloning with pronuclear and 2-cell-stage blastomeres derived from SCNT. There was no significant difference in the first cleavage rate in the 3 activation groups after SCNT. Metaphase donor cells (85.2%) had a significantly higher cleavage rate than G0/G1-phase FF with MII oocytes (70.4%) and G2-phase FF with TII oocytes (72.2%). However, reconstructed embryos were unable to develop beyond the 2-cell stage either in vitro or in vivo. Moreover, serial cloning with zygotic and parthenogenetic recipients was also unable to overcome the developmental arrest at the 2-cell-stage. To assess the cytoskeleton after SCNT, reconstructed 2-cell-stage embryos were harvested at different times after cleavage for immunostaining (anti alpha-tubulin) and mRNA abundance (beta-actin and alpha-tubulin). Reconstructed 2-cell embryos showed much thicker fibrous or disconnected MT distribution concomitant with a reduction in cytoskeletal transcripts at the late 2-cell stage. In summary, our results indicate that the developmental arrest of rat SCNT embryos was a result of abnormal MT distribution caused by improper transcription of cytoskeleton genes; however, further studies are needed to understand the early developmental block of rat SCNT-derived embryos.

36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 126
Author(s):  
V. Hall ◽  
D. Compton ◽  
P. Stojkovic ◽  
M. Nesbitt ◽  
M. Herbert ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Developmental Competence ◽  
Host Cells ◽  
Cell Stage ◽  
Double Labeling ◽  
Immunocytochemical Analysis ◽  
Parthenogenetic Activation

The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.

54 IN VITRO AND IN VIVO DEVELOPMENT OF FLAT-HEADED CAT (PRIONAILURUS PLANICEPS) CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 127
Author(s):  
A. Thongphakdee ◽  
S. Manee-in ◽  
N. Klincumhom ◽  
B. Siriaroonrat ◽  
S. Kamolnorranarth ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Individual Cell ◽  
Research Unit ◽  
Domestic Cat ◽  
Control Group ◽  
Donor Cells ◽  
Chi Square Analysis

The objectives of the study were to investigate (1) the effect of individual cell line and gender of donor cells on flat-headed cat (FC) cloned embryo production (Study I) and (2) pregnancy establishment of recipients receiving cloned FC embryos with or without domestic cat (DC) IVF embryo co-transfer. The DC IVF embryos were used as a control (Study II). Study I Three cell lines of FC fibroblasts (passage 3–5) collected from 2 females (L1 and L2; biopsied from muscle and skin, respectively) and a male (L3; biopsied from skin) were used as donor cells for nuclear transfer. Donor cells were fused with enucleated in vitro matured DC oocytes. Fused couplets were induced by electrical pulses and subsequently incubated in activation medium containing 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 4 h. Reconstructed embryos were cultured in SOFaa medium supplemented with 5% fetal bovine serum (FBS) at 38.5°C in air, and monitored for 7 days. Differences in the percentages of fusion and embryo development to a particular stage between cell lines and genders of donor cells were determined by chi-square analysis. Variations of fusion efficiency and embryo developmental success were observed between the cell lines. Greater cleavage number (P < 0.05) was observed when L1 was used as donor cells than that of L2 and L3. Developmental success to morula stage of embryo reconstructed from L1 was greater (P < 0.05) than that of L3 but not L2 (P > 0.05). However, there was no difference in the blastocyst formation success among cell lines. The development of the embryos derived from female and male donor cells at subsequent stages was not different. Study II Estrus and ovulation were induced in 15 DC recipients using 100 to 150 IU of pregnant mare serum gonadotropin (PMSG) and 100 IU of hCG (subcutaneous injection). Recipients were divided into 3 groups; (1) cloned group (n = 5) receiving FC cloned embryos (mean 41.4 ± 13), (2) co-transferred group (n = 4) receiving FC cloned and DC IVF embryos (mean 55 ± 15; 43.3 ± 15 of FC cloned and 10.8 ± 1.5 of DC IVF embryos), and (3) IVF/control group (n = 6) receiving only DC IVF embryos (mean 25 ± 9). Control DC IVF embryos were produced by co-incubation of DC oocytes with fresh DC semen for 18 h. Day 1 embryos were transferred into oviducts of recipients. Pregnancy evaluation using ultrasonography at Day 30 post-transfer demonstrated that pregnancy was not observed in any recipients in cloned group. One recipient from co-transferred group became pregnant and delivered DC IVF stillbirths (n = 2) and live kittens (n = 6). All recipients in IVF group became pregnant and 3 recipients delivered 5 DC kittens. These results indicate that (1) the individual cell line but not the gender of donor cells influences the development of FC cloned embryos and (2) with or without co-transfer of FC cloned and DC IVF embryos, FC cloned offspring was not able to be produced in the study. Table 1.Developmental success of FC cloned embryos This study was supported by the Zoological Park Organization under the Royal Patronage of H.M. the King, and the Reproductive Biotechnology Research Unit, Chulalongkorn University. A. Thongphakdee is supported by the Royal Golden Jubilee Ph.D. Program, and the Thailand Research Fund.

66 SUCCESSFUL ESTABLISHMENT OF PLURIPOTENT ntES CELL LINES FROM AGED MICE

2007 ◽  
Vol 19 (1) ◽  
pp. 151
Author(s):  
E. Mizutani ◽  
S. Kishigami ◽  
N. V. Thuan ◽  
H. Ohta ◽  
T. Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Germ Line ◽  
Chimeric Mice ◽  
Cell Nuclei ◽  
Aged Mice ◽  
Donor Cells ◽  
Tail Tip

Nuclear transfer technique has enabled us to produce cloned animals from somatic cell nuclei in various animal species to date. Moreover, it has been demonstrated that ES cell lines have been established from cloned blastocysts by somatic cell nuclear transfer (ntES cell), irrespective of sex, strains, or organs. These cells are capable of differentiating into all 3 germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. So ntES cells have gotten a lot of attention recently in the field of regenerative medicine. However, it is unclear whether ntES cells can be established from aged individuals because, in general, the cloning success rate was higher when young donor cells were used, such as fetus cells rather than adult. To answer this question, we tried to establish ntES cell lines from aged mice and then examined their pluripotency. The donor cells were obtained from tail-tip fibroblast cells of 11-month-old to 15-month-old male and female mice. After nuclear transfer, we succeeded in establishing 8 ntES cell lines from 3 aged BDF1 males and 6 ntES cell lines from 2 aged BCF1 females. The normality of these ntES cell lines was examined after passages 5 times. Karyotypes were analyzed using SKY-Fish painting, and pluripotency was examined by chimeric mice formation, in which ntES cells were injected into fertilized ICR blastocysts. As a result, most of the ntES cell lines examined had normal karyotypes, and all of the ntES cell lines tested could contribute to somatic cells of chimeric mice. Now we are examining whether these ntES cells have germ line transmission ability in chimeric mice by natural mating.

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