Embryo Cloning Research Articles

THE ROLE OF NAA, GA3, ACTIVATED CHARCOAL, AND SUCROSE ON ZYGOTIC EMBRYOS CULTURE OF HYBRID OG OPEN POLLINATED CLONE (Elaeis guineensis JACQ. X Elaeis oleifera)

Oil palm OG hybrid genetic potentials are high in term of vegetative and generative traits, such as: low height increment, compact palm, high free fatty acid and high carotenes. However, OG hybrid reproduction is hindered due to its low germination rate. So, alternative method for germination is needed, which is in vitro embryo zygotic culture. This study objective was to reveal the best medium composition for OG hybrid embryo culture by modificated several oil palm basic medium from oil palm tissue culture protocol and to understand the role of plant growth regulators, activated charcoal, and sucrose on in vitro growth and development of OG hybrid embryo. The design used was Completely Randomized Design (CRD) with 16 replication and 4 embryos for each replicates, while the treatments were: A1 = M129 (control); A2 = M129 + activated charcoal; A3 = M129+ activated charcoal + sucrose; A4 = ½ M129 + activated charcoal + sucrose; A5 = M129 + GA3 ; and A6 = M129 + GA3 + NAA. From the study we found that the best medium for OG hybrid open pollinated clone's embryo culture was M129 with GA3 and NAA (A6). This medium gave percentage of in vitro germination up to 14.06%, and later on the plantlets from this medium gave the best vegetative growth performance such as high number of leaves and primer roots.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non-human primates. The published material, especially the studies with human embryos, is controversial. Some reports suggest that twinning technology will find clinical use in reproductive medicine in the future, whereas others conclude the opposite that human twin embryos created in vitro are unsuitable not only for clinical, but also for research, purposes. The blastomere biopsy technique of embryo splitting seems to be unsuitable for either clinical or research purposes; however, embryo bisection, a preferable method of cloning in veterinary medicine, has not yet been tested on human embryos.

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

Use of peripheral blood for production of buffalo (Bubalus bubalis) embryos by handmade cloning

Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes.

Effect of Culture Medium Supplementation with b-mercaptoethanol and Amino Acid on Canine Intergeneric Embryo Development with Porcine Oocyte Cytoplasm Recipient

The present study investigated the effect of culture medium supplementation with mercaptoethanol ( ME)and amino acid (AA) on canine intergeneric embryo development with porcine oocyte cytoplasm. Porcine cumulusoocyte complexes (COCs) were collected from slaughterhouse and matured in TCM-199 supplemented with 26.2mM NaHCO, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 75 mg/l kanamycin, 10 ng/ml 3epidermal growth factor, equine chorionic gonadotropin (eCG), 10 IU/ml human chorionic gonadotropin (hCG),and 10% (v/v) porcine follicular fluid (pFF) at 39 °C in a humidified atmosphere of 5% CO for 42-44 h and donor cell 2collected from ear skin afghanhound male dog. After somatic cell nuclear transfer (SCNT), embryo developmentwere examined for cleavage rate and 144 hr for final development after cultured in media. The result shows that,amino acid and mercapoethanol addition in culture medium (NCSU-23) have no effect on embryo development.The development rate of embryo until 16 cell stage in NCSU and NCSU supplement are 4.67% and morula stage are3.73% and 4.67%.Key words : intergeneric clone embryo, canine, ( amino acid (AA)

Development of Intergeneric Canine Embryo Using Bovine and Porcine Oocyte as Cytoplasm Recipient

This study wa conducted to increase the efficiency of canine embryo production by intergeneric somatic cell nuclear transfer (SCNT) technology. The effect of oocyte recipient for development of intergeneric canine somatic cell cloning embryos were examined. Bovine and porcine cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in TCM-199 medium depend on species. Adult dog fibroblasts collected from 3.5 years old Afghandhound dog, and cell between passage 1 and passage 10 used for intergeneric somatic cell cloning using bovine and porcin oocytes as oocyte cytolplasm donor. The result showed that oocytes from bovine and porcine can de-differentiated canine nucleus and no different between bovine and porcine oocyte in fusion and embryo development in vitro. Canine intergeneric cloned embryos developed to morula stages in vitro.

37 EFFECT OF CELL MANIPULATION FOR PRODUCTION OF TRANSGENIC CELL LINES ON GOAT CLONING EFFICIENCY

The generation of transgenic cell lines through standard cell transfection/antibiotic selection procedures may have a negative effect on cell viability, which in turn may compromise SCNT cloning efficiency. The aim of this study was to evaluate goat cloning efficiency by using transfected and nontransfected and transgenic and nontransgenic somatic cells as nucleus donors. Skin fibroblast cells from 1 adult doe were subjected to transfection by electroporation with the pBC1-hGCase-Neo transgene cassette containing the human glucocerebrosidase gene sequence (hGCase), following antibiotic cell colony selection. Four distinct syngeneic donor cell types were used for cloning: (a) wild type (nontransfected, nontransgenic) control cells (C1) at low passage (P3), (b) transfected negative control (transfected, nontransgenic) cells (CT) at high passage (P8), and (c) 2 lines of transfected, transgenic cells (CA, CB) at high passages (P8 through P10). Donor cell cycles were synchronized by high confluence (&lt;95%) and 24-h serum starvation. Cloning procedures were performed by standard micromanipulation procedures. Following membrane fusion after a 1.25 kV cm–1 DC pulse for 45 µs, reconstructed structures were incubated in cytochalasin B for 1 h, and then activated in ionomycin/6-DMAP. After 12 h of IVC in G-1TM medium (Vitrolife, Englewood, CO, USA), 1-cell stage cloned embryos were surgically transferred into the oviduct of synchronous recipient females. To ascertain herd fertility and health and adequate procedures for embryo manipulation, synchronization protocols, and surgical interventions, groups of control females were subjected to cervical AI or surgical transfer of in vivo-produced 1-cell stage goat embryos (ET). Pregnancy diagnosis was performed by ultrasonography on Day 23, with weekly examinations until term. Data were analysed by the χ2 test (P &lt; 0.05), and are presented in Table 1. The transfection process and passage number did not appear to affect development, as no differences in pregnancy rates were observed between cloned groups, although results with control cells (C1 and CT) and with CA and CB lines were similar to and lower than the AI and ET groups, respectively. Loss rate after cloning was high (88.8%), which may be due to faulty reprogramming, as other procedural and biological variables involved in the cloning process were endorsed by pregnancy rates and term viable pregnancies observed in the AI and ET groups. Cloning using CA donor cells at P9 resulted in two liveborn kids, with one dying soon after birth. Both animals were confirmed by molecular analyses as hGCase transgenic clones. Table 1.Overall efficiency after AI, superovulation and embryo transfer (ET) or cloning by nuclear transfer (NT) using control (C1), sham-transfected (CT), and 2 transgenic (CA, CB) syngeneic fibroblast cells lines in goats This research was funded by FINEP.

23 IMPROVING EFFICIENCY IN WORK WITH TRANSFER OF CLONED PIG EMBRYOS

Cloning is a quite inefficient procedure with only around 10% of offspring born based on number of cloned embryos transferred. Every step to increase this level is therefore welcomed. Our group has worked with cloning of pig embryos since 2006, with the main purpose to establish a well-functioning cloning system to have transgenic piglets born as animal models for important human diseases. Here we report results from our attempts to improve efficiencies in several steps in the whole cloning procedure. Over 7.5 years, donor cells from 3 breeds were nontransgenic (50%, 4 types) or transgenic with 1 of 6 different types of gene. Oocytes from Large White (LW) sows or gilts were handmade cloned, so the zona-free cloned embryos were in vitro cultured until Day 5 to 6 to select 13 311 embryos (morulae or blastocysts) for transfer to 171 LW recipient sows or gilts. Of these, 126 were pregnant (74%; Day 35), but 20 aborted before term. A total of 704 offspring were delivered; half of the piglets were alive after 4 weeks and developed normally after that. Frequencies were compared using Chi-squared test; mean values by one-way ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Specific improvements were tested in 4 areas: (1) donor cells: stimulating reprogramming using Xenopus egg extract (Liu et al. 2013 Reprod. Fertil. Dev. http://dx.doi.org/10.1071/RD13147); (2) oocytes: preferably from sows, but also using larger gilt oocytes (Li et al. 2013 Zygote http://dx.doi.org/10.1017/S0967199412000676); (3) transfer: using both uterine sides (Theriogenology 74 : 1233); (4) number of embryos transferred: after cloning with same nontransgenic cells, embryo numbers per recipient were reduced from 90 to 30 (see Table 1). As a consequence of these different activities, overall results improved over the 7.5-year period [first 3.5 years v. last 4 years: 48% (32/67) v. 90% (94/104) recipients pregnant after transfer (P &lt; 0.05); 5.6 ± 0.6 (n = 22) v. 6.9 ± 0.5 (n = 84) piglets/litter]. In our system, one good cloning person can now produce all embryos needed for one recipient in one good working day. Transfer of fewer cloned embryos results in fewer piglets, but it reduces the workload to produce cloned embryos and does not reduce efficiency. Further work is still needed to better understand the biological and technical challenges in work with cloning; 2 important areas are quality evaluation of the donor cells used for cloning and the recipient's reaction to transfer of many embryos. In conclusion, a reasonable increase in the overall efficiency in pig cloning work was achieved, which reduces the need for personnel, time, and material when working with this technology. Table 1.Results of improving efficiency of cloning

Incidence of Abnormal Offspring from Cloning and Other Assisted Reproductive Technologies

In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.

A catalyst for change in reproductive science: John D. Biggers as a mentor’s mentor

As we have done in the past, recognizing the true founders of reproductive medicine as we know it today is both an honor and responsibility that we provide to the JARG readership. In this issue of JARG, we take pride in honoring the lifelong achievements of Professor John D. Biggers on the occasion of his 90th birthday. For us personally, and for the many trainees and colleagues who have so generously contributed to this issue, assembling an appropriate tribute for Professor Biggers is a humbling task to say the least. Consider for example the wide-ranging career accomplishments that encompass, from the very beginning, over 60 years of a tireless, critical, and collegial unselfish commitment to so many that have conspired to make reproductive biology and medicine a prominent discipline in biomedical research today. We recognize him for the significance of his contributions to reproductive biology and medicine through the lens of a subset of scientists who have graciously made the following contributions. Hardly can a single journal issue or ASRM symposium achieve a thorough and balanced accounting of the ground-breaking and persistent influences Professor Biggers has had on so many. With this backdrop, we hope the JARG readership will appreciate our humble efforts at producing a glimpse of Professor Biggers’ career from which a sense of the compleat scientist will emerge. His accomplishments at the bench are far outweighed by the lasting legacy that lives on in his mentees and by his singular efforts to bring a scholarly interpretation of the evolution of ARTs to his peers, the public, and lawmakers [1]. In fact, it is fair to say that few amongst us can claim to have made inroads into the debates surrounding IVF, embryo culture, gamete and embryo preservation and cloning to the benefit of the scientific community as a whole and society more broadly. Intent is one thing-implementation quite another! What follows is fitting testimony to his career accomplishments and impacts built from personal recollections of his colleagues and friends. Next, several papers are contributed from collaborators are complemented by a series of original papers dealing with a range of topics for which he is known and that bear directly on the purview of JARG. Biggers is well-known for his travels around the world as a representative for the World Health Organization and as an invited speaker at meetings that cross many scientific disciplines. A partial list of his role in the Society for the Study of Reproduction (SSR) includes Founding Member of SSR, 1967 (Figs. 1, ​,22 and ​and33) Fig. 1 Copy of certificate noting John D Biggers, DSc, PhD as charter member of the Society for the Study of Reproduction (1967, SSR); Biggers also served as President of SSR 1968–69, Editor-In-Chief of the Societies’ journal Biology of Reproduction ... Fig. 2 Letter received from Dr Howard W. Johnson, Jr, MD recognizing Professor Biggers on the occasion of his 90th birthday Fig. 3 Newspaper article originally published November 1, 1979 in The Ledger-Star titled “Both sides swap barbs over test-tube babies.” This article represents one of the many times Biggers expert opinion was solicited from the media during the ... SSR President, 1968–69 Editor-in-Chief, Biology of Reproduction, 1970–1974 Carl G. Hartman Award Recipient, 1986 Fitting then that we recognize his accomplishments, which cover a span of 60 years, in this special issue of JARG given the influence Biggers’ has had on the discourse, basic science discoveries, and clinical impact of his contributions to the field of human ARTs. Congratulations John and thank you for enriching so many lives!

A caffeine fix for human nuclear transfer?

Efficient human nuclear-transfer embryo cloning is here at last, prompting renewed efforts to explore hidden variables in the cloning process.

Improving development of cloned goat embryos by supplementing α-lipoic acid to oocyte in vitro maturation medium

α-Lipoic acid (LA) is a powerful antioxidant for clinical therapy of some metabolic diseases, but there are few reports about the effect of LA on animal occyte in vitro maturation (IVM). The objective of this study is to investigate the effect of supplementing LA to IVM medium on subsequently developmental competence of goat cloning embryos after somatic cell nucleus transfer (SCNT). Twenty-five micromolars LA significantly increased 12% oocyte maturation rate from control 57.8% to treated group 69.8% (P < 0.05). The reconstructed rate of cloning embryos in LA supplement group (67.3%) was significantly higher than control (56.5%, P < 0.05). Although the SCNT embryo cleavage rates did not have significant difference between the two groups (42.0% vs. 47.9%, P > 0.05), LA supplement group had significantly higher blastocyst formation rate and hatched rate than control (24.0% vs. 18.4% and 37.0% vs. 30.9%, respectively, P < 0.05). In addition, supplementing LA significantly reduced the cellular apoptosis rate of nucleus transfer blastocysts by inhibiting the expression of apoptotic activators, such as Bax, Bad, Caspase-3, and CytC genes and promoting cumulus-oocyte complexes to synthesize glutathione (GSH) and express antioxidant enzymes such as GPX4 and SOD genes. In conclusion, supplement of LA to oocyte IVM medium could improve the maturation rate and antioxidant ability of oocytes and increase the developmental competence of oocytes after SCNT.

What justifies the United States ban on federal funding for nonreproductive cloning?

This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.

The transcriptomic architecture of mouse Sertoli cell clone embryos reveals temporal–spatial-specific reprogramming

Somatic cell nuclear transfer, a technique used to generate clone embryos by transferring the nucleus of a somatic cell into an enucleated oocyte, is an excellent approach to study the reprogramming of the nuclei of differentiated cells. Here, we conducted a transcriptomic study by performing microarray analysis on single Sertoli cell nuclear transfer (SeCNT) embryos throughout preimplantation development. The extensive data collected from the oocyte to the blastocyst stage helped to identify specific genes that were incorrectly reprogrammed at each stage, thereby providing a novel perspective for understanding reprogramming progression in SeCNT embryos. This attempt provided an opportunity to discuss the possibility that ectopic gene expression could be involved in the developmental failure of SeCNT embryos. Network analysis at each stage suggested that in total, 127 networks were involved in developmental and functional disorders in SeCNT embryos. Furthermore, chromosome mapping using our time-lapse expression data highlighted temporal–spatial changes of the abnormal expression, showing the characteristic distribution of the genes on each chromosome. Thus, the present study revealed that the preimplantation development of SeCNT embryos appears normal; however, the progression of incorrect reprogramming is concealed throughout development.

“XXIst century odyssey of Medicine” stem cells and their future

The last century is undoubtedly the one of discoveries which have changed the destiny of humanity in a very short time. Quantum physics have opened the door of nanotechnology, information technologies, and their multiple applications in medicine. The discovery of penicillin and antibiotics has helped with the development of a safe and aseptic surgery. Moreover, microbiology, molecular biology, genetics, and the human genome sequencing have given us knowledge and wonderful tools to help to understand the mechanisms that rule life-sciences. They have also given us the technology to study gene transmission and their expression. Thus, it is nowadays possible to treat many diseases, for example in the field of oncology. Last, organ transplantation is part of these medical innovations. During these discoveries, especially those related to IVF, ethical problems have arisen during the cloning of embryos, attempt of embryos manipulation, and embryonic stem cells derivation for therapeutic use. Pluripotent stem cells and deriving induced pluripotent stem cells represent the greatest medical advance of this century and were awarded the Nobel Prize for Medicine in 2012 (Yamanaka, 2012). Therefore, hope has arisen in several areas like neurology, cardiology, tooth regeneration, and reproductive medicine. If it is now possible to consider repairing organs by autologous grafting like after myocardial infarct, or spinal cord injury (Hibaoui and Feki, 2012), the approach using pluripotent stem cells in reproductive medicine raised ethical and scientific questions. Obtaining functional sperm or oocytes from skin derived-iPS cells may lead to the disappearance of the term of infertility, and IVF programs would benefit from this development. Stem cell field also offers other possibilities like for pharmacology of drug testing, disease modeling, and the discovery of new gene functionalities. The reviews of this special issue treated several of the aforementioned fields. This century will be the one of stem cell biology and genetics: the humankind will be attracted by findings of this science. It will not create but will try to repair, to live longer, and to rejuvenate. If Einstein believed that genius is intuition, we have enough models at our disposal to take over the following meaningful sentence of Master Eckhard who said: “it is not our actions that sanctify us, but, we who sanctify our actions.”

“XXIst century odyssey of Medicine” stem cells and their future

EDITORIAL article Front. Physiol., 10 September 2013Sec. Craniofacial Biology and Dental Research https://doi.org/10.3389/fphys.2013.00250

Valproic acid improved in vitro development of pig cloning embryos but did not improve survival of cloned pigs to adulthood

The objective was to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro and in vivo development of Wuzhishan miniature pig somatic cell nuclear transfer (SCNT) embryos. Experiment 1 compared in vitro developmental competence of nuclear transfer embryos treated with various concentrations of VPA for 24 h. Embryos treated with 2 mM VPA for 24 h had a greater rate of blastocyst formation compared with control or embryos treated with 4 or 8 mM VPA (21.5% vs. 10.5%, 12.6%, and 17.2%, P < 0.05). Experiment 2 examined the in vitro developmental competence of nuclear transfer embryos treated with 2 mM VPA for various intervals after chemical activation. Embryos treated for 24 h had higher rates of blastocyst formation than the control or those treated for 4 or 48 h (20.7% vs. 9.2%, 12.1%, and 9.1%, P < 0.05). In Experiment 3, an average of 207 (range, 192–216) nuclear transfer embryos from the VPA-treated group were transferred to surrogate mothers, resulting in three pregnancies. Two of the surrogates delivered a total of 11 live piglets. However, for unknown reasons, nine of 11 piglets in the VPA-treated group died within 1 to 5 d after birth. Untreated control embryos (average, 205; range, 179–225) transferred to four surrogate mothers resulted in three pregnancies, two of which delivered a total of 12 live offspring, although four of 12 piglets in the VPA-untreated group died (cause unknown) within 1 to 3 d, whereas eight of the 12 piglets in the VPA-untreated group survived more than 3 or 4 mo. The average birth weight of the two litters from the VPA-treated group tended (P < 0.05) to be lower than that from the control groups (551.6 g vs. 675.2 g). In conclusion, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2% vs. 67.0%; P < 0.05).

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.

In Vitro Embryo Production in Buffalo: Basic Concepts

In vitro embryo production involves a combination of the techniques of in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes. The in vitro matured oocytes are incubated with suitably processed spermatozoa for an appropriate period of time for carrying out IVF. The presumptive zygotes are then cultured in vitro up to the blastocyst stage at which these could either be transferred to synchronized recipients for producing live offspring or cryopreserved for future use. The IVM, IVF and IVC are also integral parts of a number of other reproductive technologies like embryo or somatic cell cloning, production of transgenic animals or production of embryonic stem cells etc., which are expected to bring about a quantum jump in the population of superior genetic merit buffaloes or be useful to mankind in other ways.