Process Of Somatic Cell Nuclear Transfer Research Articles

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor.

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 μM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998-2010).

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000 + ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

Robotic Batch Somatic Cell Nuclear Transfer Based on Microfluidic Groove

Somatic cell nuclear transfer (SCNT), which is an important procedure in cloning, has been conducted manually for decades. The operating efficiency drops sharply in batch SCNT because of the long-time observation under microscopy and the time-wasting traditional process. Though the operating time was reduced by robotic SCNT in previous studies, the traditional operating process was still used. In this article, we designed a new robotic batch SCNT process based on a microfluidic groove and two micropipettes in parallel. By using this new SCNT process, the operating area switching, objective lens conversing, and focusing on traditional SCNT process were eliminated, and oocyte localization was simplified, which saved much operating time. Experimental results showed that the new robotic batch process reduced about 50 s (41.7%) compared with the manual process (proposed 70 s versus manual 120 s). A success rate of 93.3% ( ${n} =30$ ) and a survival rate of 96.4% were achieved ( ${n}=28$ ), which were similar to manual process. The new robotic batch SCNT method demonstrated a high degree of efficiency and reproducibility. Note to Practitioners —This article presented a new robotic somatic cell nuclear transfer (SCNT) process. This new robotic SCNT process introduced a microfluidic groove for oocyte storage and two micropipettes for oocyte enucleation and oocyte injection, respectively. We save much operating time since the operating area switching, objective lens conversing, and focusing on traditional SCNT process were eliminated, and oocyte localization was simplified. Experimental results have demonstrated the efficiency and reproducibility of the new robotic SCNT process. This new robotic SCNT process has great potential for many other applications, e.g., ICSI, embryo microinjection, and cell biopsy. Commercialization of the proposed technology may lead to the improvement in SCNT industry. In current experiments, the somatic cells sometimes were injected at the same time, which led to the failure of the experiments. In the future, we will apply control algorithms to control the motion of multiple cells.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p<.05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p<.05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p<.05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

Dynamic characteristics of the mitochondrial genome in SCNT pigs.

Most animals generated by somatic cell nuclear transfer (SCNT) are heteroplasmic; inheriting mitochondrial genetics from both donor cells and recipient oocytes. However, the mitochondrial genome and functional mitochondrial gene expression in SCNT animals are rarely studied. Here, we report the production of SCNT pigs to study introduction, segregation, persistence and heritability of mitochondrial DNA transfer during the SCNT process. Porcine embryonic fibroblast cells from male and female Xiang pigs were transferred into enucleated oocytes from Yorkshire or Landrace pigs. Ear biopsies and blood samples from SCNT-derived pigs were analyzed to characterize the mitochondrial genome haplotypes and the degree of mtDNA heteroplasmy. Presence of nuclear donor mtDNA was less than 5% or undetectable in ear biopsies and blood samples in the majority of SCNT-derived pigs. Yet, nuclear donor mtDNA abundance in 14 tissues in F0 boars was as high as 95%. Additionally, mtDNA haplotypes influenced mitochondrial respiration capacity in F0 fibroblast cells. Our results indicate that the haplotypes of recipient oocyte mtDNA can influence mitochondrial function. This leads us to hypothesize that subtle developmental influences from SCNT-derived heteroplasmy can be targeted when using donor and recipient mitochondrial populations from breeds of swine with limited evolutionary divergence.

Analysis of Endoplasmic Reticulum (ER) Stress Induced during Somatic Cell Nuclear Transfer (SCNT) Process in Porcine SCNT Embryos.

ABSTRACTThis study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

Comparison of Developmental Efficiency of Murine Somatic Cell Nuclear Transfer Protocol

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher’s competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

Faithful expression of imprinted genes in donor cells of SCNT cloned pigs

To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P-PEF (parthenogenetic-PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C-PEF (cloned-PEF), P-PEF vs. CP-PEF (cloned-P-PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.

On Clone as Genetic Copy: Critique of a Metaphor

A common feature of scientific and ethical debates is that clones are generally described and understood as “copies” or, more specifically defined, as “genetic copies.” The attempt of this paper is to question this widespread definition. It first argues that the terminology of “clone as copy” can only be understood as a metaphor, and therefore, a clone is not a “genetic copy” in a strict literal sense, but in a figurative one. Second, the copy metaphor has a normative component that is problematic in the context of descriptive science and may support or indicate the ethically relevant phenomenon of objectification of animals. In order to support the argument against the common conception of a clone as a copy, the biotechnological principles of somatic cell nuclear transfer (SCNT) cloning will be examined. On this basis, it will be shown that the metaphor is valid because of similarities between the phenotype, the genotype, or the nuclear DNA sequence of the clone and its progenitor by using three prominent levels of comparison (clone as phenotypical, genotypical, and nuclear copy). Focusing on the process of SCNT, it will be shown that cloning as copying or doubling has to be redefined for scientific purposes because it is neither necessary nor does it fit to the biotechnological principles of cloning. It is more accurate to understand SCNT cloning as a process of splitting rather than of doubling or copying. In the second part, a deconstructivist analysis based on Jacques Derrida’s description in Positions (1981) will reveal the normative potential of the original–copy dichotomy. I will be showing that it includes an asymmetrical power structure between the original (progenitor) and the copy (clone) and that this structure can be reversed or at least considered unstable. Therefore, arguments that build on that metaphor must be reconsidered. Moreover, the analysis reveals that applying a terminology to humans and animals that is commonly used for things becomes the language of objectification. Two selected examples, fungibility and violability, based on Martha Nussbaum’s notion of objectification will support the thesis of objectification, display its normative consequences, and put the clone as a copy metaphor in a broader range of ethically questionable research tendencies.

A Novel Method of Somatic Cell Nuclear Transfer with Minimum Equipment.

Somatic cell nuclear transfer (SCNT) is an exceptional experimental biology technique with an arguably great contribution to our current understanding of developmental plasticity. Many students and young researchers are interested in taking advantage of SCNT virtues in their experiments but the cost of micromanipulation microscopes, intensive training programs, and also the sophisticated process of SCNT may dissuade them from entering this amazing field of science. Here, we describe the details of a streamlined manual method of SCNT that can be performed using very basic equipment found in every embryology laboratory: the Pasteur pipette and stereomicroscope. The overall method introduced is very simple and a person with no previous experience in cloning can learn and adopt the basic routines of this technique independently.

X-Linked Gene Transcription Patterns in Female and Male In Vivo, In Vitro and Cloned Porcine Individual Blastocysts

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects.

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

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

34 MEIOTIC RECOMBINATION IN SOMATIC CELL NUCLEAR TRANSFER BULLS AND THEIR OFFSPRING

In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) in the livestock industry, these vital steps have begun to be bypassed. Despite this, there have been few studies carried out on the reproductive characteristics of cloned animals and none to date regarding the consequences on the meiotic process. As these procedures grow in popularity and use, the importance of evaluating the long-term viability, health, and productivity of cloned animals and any subsequent offspring in future generations to validate potential applications of the technology is increasingly evident. Previously, studies of recombination and synapsis have focused on the physical observation of chiasmata formation in meiotic chromosomes; however, in recent years, the characterization of proteins that localize to the sites of crossing-over and of proteins present in the synaptonemal complex have permitted the study of meiotic recombination using a precise direct immunocytogenetic approach. Cytological analysis of meiotic cells obtained from the testicular tissue of five normal bulls of proven fertility, two SCNT transgenic bulls (Powell et al. 2004 Biol. Reprod. 71, 210–216, 2004), and four reproductively mature offspring of SCNT bulls was performed in order to detect the effects of SCNT on the meiotic process. Over 50 pachytene cells per animal were analyzed by immunofluorescence using antibodies against the synaptonemal complex protein 3 (SCP3) and the mismatch repair protein 1 (MLH1) located on the crossover sites. Data were analyzed using a mixed model analysis of variance with repeated measurements to determine group effects (SAS 9.1; SAS Institute, Inc., Cary, NC, USA). The average number of crossovers per spermatocyte for the non-SCNT bulls (42 � 4 SD, min: 33, max: 56), SCNT bulls (43 � 5 SD, min: 35, max: 56), and SCNT offspring (46 � 4 SD, min: 37, max: 58) were quite similar among the cells of the same individual; however, inter-individual variation was observed. These results are the first documentation of the normal range of variability of recombination distribution within the cattle genome and suggest that the SCNT process does not affect meiotic recombination. This work was funded by NSERC and the CRC program.

Human therapeutic cloning (NTSC)

Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.