Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation,colchicine treatment and gene transfection. Results are as follows: ( Ⅰ ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3%vs. 4.9%, P＜0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25 μm) as donor nuclei was higher than that from large cells (25-33 μm) and small cells (8-15 μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS( 11.8% vs. 18.6%, P＞0.05). (Ⅳ) Fetal fibroblasts treated with 0.05 μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P＞0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P＜0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05 μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower     

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho⁺-Hd⁻) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho⁻-Hd⁺) or non-heated (Ho⁻-Hd⁻) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6-32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9-33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.     

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.     

The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos

Two experiments, one comparing nuclear transfer (NT) embryo activation compounds, the other donor cell treatments, were conducted with a goal of identifying factors that improve the in vitro development of cloned bovine embryos. In experiment 1, 539 NT embryos were produced by combining serum starved bovine fetal fibroblasts with enucleated in vitro matured oocytes, activated with ionomycin, then randomly allocated to be incubated for 4 hours in either Butyrolactone-I (BL-I) or 6-dimethylaminopurine (DMAP). There was no significant difference in development to blastocyst or compact morula of fused embryos at Day 6.5 between BL-I and DMAP activated embryos (22.4% vs. 20.2%; p = 0.18). Karyotyping of 20 blastocysts and compact morula from each group determined that 65% of BL1 and 63% of DMAP embryos were diploid with the remainder mixoploid (2n + 4n). In Experiment 2, the development of 389 NT embryos reconstructed from either serum starved or serum fed fetal fibroblasts was assessed. More Day 7 blastocysts and compact morula developed in the serum starved group (34.5% vs. 18.8%; p = 0.008). To verify the viability of BL-I activated embryos, 10 blastocytes from experiment 2 were transferred into 4 recipient cows. Two morphologically normal fetuses, genetically identical to the original fetal cell line, were surgically recovered at day 45 of gestation. In summary, serum starvation of bovine fetal fibroblasts prior to NT significantly improved development to blastocyst. Additionally, we have shown that BL-I is a novel alternative compound for use in combination with ionomycin to activate NT embryos.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (45.7% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36h to 42h (83.6% vs. 96.7%, p<0.05). However, no statistical difference were observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.8%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT), combined with genome modification techniques, is a very pow-erful tool for agriculture, medicine and fundamental research on basic biological mechanisms. The effi-ciency of producing transgenic animals is greatly prom…     

Development of a zona-free method of nuclear transfer in the mouse

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (45.7% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36h to 42h (83.6% vs. 96.7%, p<0.05). However, no statistical difference were observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.8%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p<0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Differential expression pattern of key regulatory developmental genes in pre-implant zona free cloned vs in vitro fertilized goat embryos

The success of Somatic cell nuclear transfer (SCNT) primarily depends on the extent of reprogramming of donor cells genome. The error of reprogramming may lead to inappropriate expression of embryonic genes at any stage of development. Under the present study the relative expression of different genes related to pluripotency (Oct-4 and Nanog), growth factors (IGF-2 and IGF-2R) and DNA methyltransferase gene (Dnmt-1) was evaluated in SCNT embryos at 8–16 cells, morula and blastocyst stages as compared to IVF group. In SCNT, significantly higher degree of relative expression was observed for Oct-4 in morula (1.41) and blastocysts (1.14) as compared to 8–16 cells (referral stage) whereas in IVF, a lower expression was observed at morula (0.82) stage. The expression of Nanog in SCNT embryos was increased significantly in morula (2.23) and decreased subsequently in blastocyst (0.56), whereas it was increased significantly from 8 to 16 cells to morula (1.62) and blastocyst (4.5) of IVF group. The IGF-2 and IGF-2R showed significantly higher expression rates in morula and blastocysts of SCNT (6.56, 5.90 and 1.11, 1.4) and IVF (8.69, 8.25 and 2.96, 3.91) embryos, respectively as compared to referral stage. The expression of Dnmt-1 was significantly higher in SCNT morula (1.29) and blastocyst (1.15) however in IVF, it was similar in 8–16 cells stage and morula but, higher in blastocyst (1.58). The dissimilar pattern of gene expression of SCNT might be a consequence of incomplete reprogramming of donor nucleus which resulted into lower blastocyst rate of SCNT as compared to IVF embryos.     

Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos

The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups—contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods—one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

体细胞的组织来源及培养代数对猪核移植重构胚发育的影响

本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后,将经血清饥饿(0．5?s)培养2-9d、0．1mg/L Aphidicolin (APD)培养 0．5?S培养2-9d或一般培养法(10?S)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞,直接注射到去核的卵母细胞质中,或注射到卵周隙中。再经电融合(100V/mm,30μs,电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6- DMAP激活处理,体外培养6天。耳皮成纤维细胞和颗粒细胞经0．1mg/L APD 0．5?S培养处理后的重组胚卵裂率,均高于血清饥饿和一般培养处理的同种供体细胞(P＜0．01)。卵丘细胞、颗粒细胞经0．1mg/L APD 0．5?S处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P＜0．05)。以猪颗粒细胞为核供体时,电融合法的重构胚分裂率显著高于胞质内注入法(P＜0．05),但囊胚发育率无显著差异(P＞0．05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞,其具有正常二倍染色体的细胞比例均无显著差异(P＞0．05);以这2种细胞不同培养代数做供体进行核移植时,各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P＞0．05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定;(2)0．1mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果,血清饥饿培养则无明显效果;(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞．核移植后都能得到体细胞克隆的囊胚,但前者的效果略优于后者,且其核移植效果不受供核细胞培养代数的影响;(4)电融合核移植胚胎的发育率高于胞质内直接注入法,但两者的总体效率相近。     

Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning, which is based on human somatic cell nuclear transfer, is one of our major research objectives. Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos, the effects of type, passage, and preparation method of donor cells on embryo development remain unclear. In our experiment, cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell, skin fibroblast, and cumulus cells. The cumulus cell embryos showed significantly higher development rates than the other two (P < 0.05). The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference. Also, fluorescence in situ hybridization (FISH) was conducted to detect nuclear derivation of the embryos. The result showed that the nuclei of the inter-species cloned embryo cells came from human. We conclude that (1) cloned embryos can be constructed through human-rabbit interspecies nuclear transfer; (2) different kinds of somatic cells result in different efficiency of nuclear transfer, while in vitro passage of the donor does not influence embryo development; (3) refrigeration is a convenient and efficient donor cell preparation method. Finally, it is feasible to detect DNA genotype through FISH. Translated from Zoological Research, 2005, 26(4): 416–421 [译自: 动物学研究]     

Intergeneric somatic cell nucleus transfer in marbled cat and flat-headed cat

Intergeneric nucleus transfer (ig-NT) is a promising technique to produce offspring of endangered species. The objectives of this study were to (1) investigate the in vitro development of marbled cat (MC; Pardofelis marmorata) and flat-headed cat (FC; Prionailurus planiceps) ig-NT embryos reconstructed from domestic cat (DC; Felis catus) oocytes (Experiment 1), (2) evaluate the effect of individual FC donor cell lines on NT success (Experiment 2), and (3) assess the developmental ability of FC-cloned and DC-IVF embryos in vitro and in vivo after oviductal transfer (Experiment 3). In Experiment 1, the morula rate of FC-reconstructed embryos was significantly higher than those of MC and DC embryos but lower than that of parthenogenic DC embryos. However, blastocyst rate was not different. In Experiment 2, FC-ig-NT embryos reconstructed from female muscular tissue had significantly higher morula rate in comparison with those derived from other donor cell lines. However, there was no difference in blastocyst rate among cell lines. In Experiment 3, in vitro development of FC-ig-NT embryos was lower than that of DC-IVF embryos. The competency of in vivo development of FC-ig-NT and/or DC-IVF embryos was investigated by assessing pregnancy rate after their transfer into DC recipients. Domestic cat recipients receiving only FC-ig-NT embryos, FC-ig-NT embryos in one side of the oviduct and DC-IVF embryos contralaterally (co-transfer), and only DC-IVF embryos were observed. No pregnancy was detected in all recipients receiving FC-ig-NT embryos. One recipient receiving co-transferred embryos became pregnant, then delivered DC-IVF dead fetuses (n = 2) and live kittens (n = 6). All recipients receiving DC-IVF embryos became pregnant, and three of six recipients delivered five DC-IVF kittens. These results illustrate the developmental capacity of MC- and FC-ig-NT embryos up to the blastocyst stage. Individual donor cell line affects the developmental success up to the morula stage of FC-ig-NT embryos. Improving the developmental competence and quality of FC-ig-NT embryos may be required for implantation and development to term of FC-ig-NT offspring.