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.

Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set.

BACKGROUND: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. RESULTS: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. CONCLUSION: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Survival of embryos and calves derived from somatic cell nuclear transfer in cattle: a nationwide survey in Japan.

To obtain data concerning the survival of embryos and calves derived from somatic cell nuclear transfer (SCNT) in Japan, a nationwide survey was carried out in April, 2009. As a result, data concerning 3264 embryo transfers (ETs) with SCNT embryos which produced 301 calves were accumulated and their survival was analyzed. The present survey revealed that survival rates of transferred bovine embryos and produced calves derived from SCNT had not improved over a decade (1998-2007). A remarkable feature of the pregnancies with SCNT embryos was a high incidence of spontaneous abortions. When the decade was divided by the occurrence of bovine spongiform encephalopathy (BSE) in 2001, significant decreases in the 'after BSE' period (2002-2007) were observed in the percentages of calves born (P<0.01), calves living at birth (P<0.05), calves living for 24 h (P<0.05) and 6 months (P<0.01). Abortions that occurred during 61-99 days after ETs were significantly increased (P<0.01) in the 'after BSE' period. Certain kinds of regeneration that occurred in oocytes during the 15-20 h of storage of bovine ovaries at 10-15 °C as a part of BSE inspection might have had some negative effects on SCNT embryos when these oocytes were used as recipients of SCNT.

Acetylation level of histone H3 in early embryonic stages affects subsequent development of miniature pig somatic cell nuclear transfer embryos.

Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into an embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of histone on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 +/- 2.7 and 56.6 +/- 2.7 vs. 43.5 +/- 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitro- fertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs.