Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell

The present study was conducted to establish an efficient production system for bovine transgenic somatic cell nuclear transfer (SCNT) embryos, the effect of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos were examined with their expression rates of a marker gene. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6, as a carrier. In Experiment 1, three types of bovine cells were transfected at passages 2 to 4, and then trypsinized and GFP-expressing cells were randomly selected and used for SCNT. Developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In all cell types used, GFP expression rates of SCNT embryos gradually decreased with the progression of embryo development. In Experiment 2, the effect of passage number of cumulus cells in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed, but significantly higher GFP expression was shown in blastocysts reconstructed with cumulus cells at early passage. In Experiment 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 microm) or small cell (<30 microm)] at passages 2 to 4 were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells as well as fetal cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.     

Effect of fusion/activation protocol on in vitro development of porcine nuclear transfer embryos constructed with foreign gene-transfected fetal fibroblasts

The effect of fusion/activation protocol on in vitro development of porcine nuclear transfer (NT) embryos constructed with foreign gene-transfected somatic cells were investigated. NT embryos were produced by using enucleated M II oocytes and enhanced green fluorescence protein (EGFP) gene-transfected or non-transfected porcine fetal fibroblasts. One group of NT embryos received a single electrical pulse to induce fusion and activation simultaneously (FAS). The other group was fused 2 hr before activation (FBA) using two kinds of electrical pulses. Electrically activated NT embryos in both groups were treated with cycloheximide (CHX) before culture to assess the development to the blastocyst stage. After 6 days of culture, all morulae and blastocysts derived from EGFP-transfected fibroblasts emitted green fluorescence without mosaicism, and EGFP-gene product was also detected in all morulae and blastocysts examined. NT embryos undergoing FAS showed higher developmental capacity to blastocysts than those undergoing FBA, regardless of the EGFP transfection into the nuclear donor cells. The results also indicated that EGFP-gene transfection into nuclear donor cells has no obvious deleterious effect on the development of NT embryos to blastocysts.     

Production of transgenic bovine cloned embryos using piggybac transposition

Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.     

Generation of Buffalo (Bubalus bubalis) Transgenic Chimeric and Nuclear Transfer Embryos Using Embryonic Germ-Like Cells Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)‐like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG‐like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE‐EGFP‐IRES‐Neo‐dNdB) using Lipofectamine^TM 2000 and selected by culturing in 200 μg/ml G418 for 6–8 days. G418 resistant fibroblasts and EG‐like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8–16 cells embryos were injected with EG‐like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG‐like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG‐like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG‐like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG‐like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG‐like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.     

供体细胞培养处理方法对水牛核移植效果的影响

以经常规培养法 (DMEM 10 % FCS)、血清饥饿法 (DMEM 0 .5 % FCS培养 5～ 10 d)和 Apidicolin- APD结合血清饥饿法 (0 .1mg/ L APD培养 2 4 h,DMEM 0 .5 % FCS培养 1～ 18d)培养处理的水牛卵巢颗粒细胞和水牛成体耳部成纤维细胞作供核 ,分别采取带下注核法和胞质内注核法进行核移植。同一供核细胞各处理组间的核移植胚融合率 (以颗粒细胞作供核 )以及重组胚的囊胚发育率无明显差异 (P>0 .0 5 ) ,但经 APD 0 .5 % FCS培养处理供体细胞核移植后的分裂率显著高于其他组 (P<0 .0 5 )。用 7%乙醇处理的成体耳部成纤维细胞进行核移植 ,其重组胚的分裂率和囊胚发育率与对照组 (不含乙醇 )均无明显差异 (P>0 .0 5 )。结果表明 ,(1)血清饥饿处理水牛供体细胞对其核移植效果没有影响 ;(2 ) DNA合成抑制剂 APD结合血清饥饿培养处理水牛颗粒细胞和成体耳部成纤维细胞 ,可提高其核移植效果 ;(3)乙醇预激活处理水牛成体耳部成纤维细胞 ,对其核移植效果没有影响     

Selection of donor nuclei in somatic cell-mediated gene transfer using a co-transfection method

In this study, we introduced a co-transfection method for the selection of donor nuclei in somatic cell-mediated nuclear transfer. Two vectors were constructed in our experiment. One was pMSCV-GFP carrying the neomycin-resistant gene (Neo(r)) and the green fluorescent protein (GFP) reporter gene; the other was pBC1-GFP carrying the mammary gland-specific promoter and target gene GFP. Ovine adult fibroblasts were co-transfected with pMSCV-GFP and pBC1-GFP. The data from this work demonstrated that the GFP genes in both vectors could successfully co-integrate into the genomes of ovine adult fibroblasts in three of the four transgenic cell clones assayed. Furthermore, PCR analysis of transgenic embryos proved that the GFP genes in both vectors could co-integrate into the genomes of the reconstructed embryos. Subsequently, analysis of the developmental rate of the reconstructed embryos after nuclear transfer indicated that the blastocyst rate from the co-transfected donor cells was similar (approximate 8 percent) to that from individual pMSCV-GFP transfected donor cells. The influence of co-transfection resulting in modification of donor nuclei on development of reconstructed embryos was also investigated. The results of flow cytometric analysis indicated that the co-transfected ovine fibroblasts had similar quiescent characteristics in terms of cell cycle (G0+G1 percent: 73.20 +/- 4.04) to the individual pMSCV-GFP transfected fibroblasts (G0+G1 percent: 70.77 +/- 1.19) after they were treated with serum starvation for five days. Our results suggest that the co-transfection method can be used for selection of donor cell clones in somatic cell-mediated gene transfer experiments. It can be potentially extended to applications related to expression of functional protein in mammary glands and other transgenic research relevant to nuclear transfer.     

Production of blastocysts after intergeneric nuclear transfer of goral (Naemorhedus goral) somatic cells into bovine oocytes

Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.     

转GFP基因核移植牛胚胎的研究

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.     

Effects of downregulating DNA methyltransferase 1 transcript by RNA interference on DNA methylation status of the satellite I region and in vitro development of bovine somatic cell nuclear transfer embryos

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.     

Timing and uniformity of embryonic gene activation affect subsequent pre-implantation development of cloned bovine embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.     

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.     

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.     

In vitro developmental potential of bovine nuclear transfer embryos derived from primary cultured cumulus cells

The in vitro development and the quality of blastocysts produced from the nuclear transfer (NT) embryos reconstituted from primary cultured cumulus cells (NT-cumulus) were examined compared to in vitro fertilized embryos (IVF) and NT embryos reconstituted from the embryonic blastomeres (NT-blastomere). The cleavage rate, and the development to blastocyst were the same for all three sets of embryos. The time required for blastocoel formation starting from the time of the initial cleavage was shorter for NT embryo groups than IVF ones. All experimental groups produced morphologically similar and normal blastocysts containing the same cell number. The percentage of the blastocysts with normal chromosomal complements were the same for NT-cumulus and IVF.     

核移植前激活受体卵子对牛核移植卵体外发育的影响

本研究探讨了核移植前对受体卵子进行激活、细胞融合开始时间及供体受精卵细胞周期调节对核移植卵体外发育的影响。其结果显示核移植前对受体卵子激活组的细胞融合率与对照组没有差异 ,但重组胚胎的卵裂率、8～ 16细胞期胚胎及囊胚的发育率比对照组明显提高 ;核移植前激活的受体卵子分别在卵子体外成熟开始的第30h和 4 5h与供体细胞进行细胞融合 ,结果 ,30h组的细胞融合率和卵裂率与 4 5h组没有差异 ,但发育到 8～ 16细胞期及囊胚的发育率均比 4 5h的高 ;将供体受精卵用诺考达唑 (Nocodazole)处理后 ,进行核移植的结果 ,处理组的细胞融合率、卵裂率、发育到 8～ 16细胞期和囊胚的发育率与对照组无差异     

Nuclear transfer in cattle : effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes

The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.