Reconstruction of nuclear transfer embryos in goats and cattle [electronic resource]

The low survival rates of nuclear transfer fetuses and neonates in goats and cattle have been linked to placental abnormalities. A series of studies was designed to investigate the possibility of supplementing nuclear transfer embryos with electrofused embryos to generate placental tissue in goats and cattle. The initial study was designed to determine if the breeding season of goats could be extended with hCG treatment. Progesterone concentrations in treated does increased but pregnancy rates were unaffected. In the second study, goat embryos were electrofused and combined with nuclear transfer embryos at the 8-cell stage to produce the first offspring as a result of electrofused embryo complementation in goats. The remainder of the studies focused on electrofused embryos in cattle. The method of electrofusion was studied and it was determined that fusion efficiency and developmental rates after two fusogenic pulses were not different from fusion efficiency and developmental rates after a single pulse. The latter study also showed that the time of cleavage following in vitro fertilization affected the cleavage and blastocyst rates of embryos after electrofusion. In the next study, electrofused embryos were aggregated with nuclear transfer embryos at the 8-cell stage. Aggregate embryos developed to the blastocyst stage at the same rate as electrofused and nuclear transfer control embryos. The final study was a series of experiments conducted to characterize the nuclear status of electrofused embryos. In the first and second experiments of the series, embryos were stained following electrofusion and it was found that more embryos were tetraploid and fewer were binucleate when embryos were electrofused later after cleavage. The third and fourth experiments in this series examined the stage of the cell cycle prior to electrofusion. These experiments indicated that the embryos electrofused at 30 hours post-insemination were in the G2 phase of the cell cycle. It was concluded that the stage of the cell cycle would be an important factor in the production of tetraploid embryos via electrofusion and this should be the basis of future research in this area

Establishment of pluripotent cell lines from vertebrate species - Present status and future prospects

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species, However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse a nd chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals. Copyright (C) 1999 S. Karger AG, Basel

Cloning in action: can embryo splitting, induced pluripotency and somatic cell nuclear transfer contribute to endangered species conservation?

The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs

Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs

THE EVOLVING PICTURE IN OBTAINING GENETICALLY MODIFIED LIVESTOCK

Animais de produção geneticamente modificados são aqueles que tiveram uma sequência de DNA endógena modificada ou um DNA exógeno introduzido em seu genoma. Animais de produção geneticamente modificados apresentam grande potencial como modelo para estudo de doenças humanas, para produção mais eficiente de carne e derivados do leite, para xenotransplante e para produção de produtos sob grande demanda para saúde humana. Diferentes abordagens têm sido descritas para obtenção de animais de produção geneticamente modificados, as quais apresentam eficiências, vantagens e limitações variáveis. O objetivo da revisão é descrever o histórico da obtenção de animais de produção geneticamente modificados, os principais obstáculos, abordagens atuais e perspectiva futuras sobre a tecnologia

Investigating the consequences of chromosome abnormalities arising during pre-implantation development of the mouse

The majority of human pre-implantation embryos created through in vitro fertilization (IVF) are mosaic as they are constituted of a mixture of diploid and aneuploid cells. Chromosome abnormalities are widely believed to contribute towards the relatively low success rates of IVF treatment. Consequently major efforts have been undertaken to develop effective tools to aid the selection of embryos with minimal abnormalities with the aim of improving clinical outcomes. However, the ultimate fate of mosaic embryos is not known. Human embryo research is limited by practical and ethical constraints, and directly relevant animal studies are sparse. To circumvent many of these limitations, a mouse model for pre-implantation chromosome mosaicism was developed. Acute chromosome segregation errors were induced in cleavage stage mouse blastomeres by bypassing the spindle assembly checkpoint (SAC). This model was used to investigate the fate of abnormal cells within the developing pre-implantation embryo, and the ultimate developmental outcome of mosaic embryos. Time-lapse imaging of pre-implantation development revealed that cells with chromosome abnormalities were progressively depleted during blastocyst maturation; inner cell mass (ICM) cells exhibited higher rates of apoptosis, while in the trophectoderm (TE) lineage effects on the cell-cycle predominated. Depletion continued throughout post-implantation development. Significantly, the presence of a critical number of control blastomeres within the embryo could rescue the early post-implantation lethality that occurred in embryos containing high rates of abnormalities. Thus it was demonstrated that mosaic embryos can achieve full developmental potential and that abnormal cells are progressively depleted as development proceeds. Finally, the mechanisms responsible for eliminating the abnormal cells from the embryo were investigated, revealing that embryos containing chromosome abnormalities may have increased metabolic requirements which could contribute to their clonal depletion; a feature previously characterised in aneuploid cells in the context of cancer research.This work was sponsored by a Wellcome Trust Clinical PhD Fellowshi

The effect of age and sex on the growth patterns of bovine cell lines

The influence of donor animal sex or age on in vitro bovine cell culture was evaluated to provide foundation information for the selection of donor tissue for nuclear transfer. Skin biopsies were taken from each of sixteen individuals including four bulls (B), four cows (C), four male calves (MC), four female calves (FC). At passage 2, cells from in vitro culture of cell lines were influenced not by gender but by age in the mean cell generation time (MGT). When evaluating familial lineage, comparison between related and unrelated groups showed that most comparisons do not show significant differences in lag time, stationary phase viable cell counts (SPCC) and MGT. In each cell line, there was high cell viability throughout the growth curves, indicating stable cell maintenance and proper cell harvest was conducted in this study. At passage 4, MGT of each cell line was not influenced by age but by sex at passage 2, however, at a later cell passage (by passage 4), the MGT of each cell line was not affected by either sex or age of the donor. By passage 4, the MGT of each cell line was not affected by either sex or age. As passages continued, the extrinsic environmental factors likely influenced the MGT. Cell cycle analysis at passage 4 on day 0 of this study showed that \u3e 90% of cells were in G0/G1 portion in each cell line of all groups. Cell lines from younger donors were more frequently at higher G0/G1 percentages, or synchronized than those derived from older donors. Thus, age of donor animal could be a factor in selecting cell line for NT, especially when G0/G1 nuclei are intended for use. Male groups (B and MC) showed higher stationary phase viable cell counts than female groups (C and FC). Most comparisons showed no significant differences in lag time, SPCC and MGT between related and unrelated familial lineage groups. Each cell line showed constant viability (94.36 to 97.98%) at passage 4 throughout the growth curves

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models

Mechanisms behind the fate of early chromosomal and transcriptional heterogeneities in the mouse embryo

A series of events during the first four days of mouse embryo development leads to the formation of a blastocyst. The blastocyst consists of neatly segregated three lineages: the epiblast (EPI), which will form the fetus, the extra-embryonic primitive endoderm and the outer layer of the extra-embryonic trophectoderm (TE). This organization prepares the embryo for implantation and subsequent development. This study aims to explore two broad questions: 1) what mechanisms dictate the fate of the progenies of the chromosomally abnormal cells generated during the 4-8 cell stage division; 2) how the transcriptional heterogeneities between the blastomeres of the 4-cell stage embryo affect subsequent lineage segregation. A high incidence of aneuploidy in the early cleavage divisions is considered the principal cause for low human fecundity and developmental defects. However, there is a dramatic decline in the prevalence of aneuploidy as gestation progresses. To understand the fate of aneuploid cells, a mouse model of chromosome mosaicism was used. In vitro culture system and live imaging demonstrated that aneuploid cells were eliminated from the EPI by apoptosis both during pre- and peri-implantation development. Also, aneuploid cells displayed chronic proteotoxic stress. Subsequently, p53-mediated autophagy eliminated aneuploid cells from the EPI. Unlike aneuploid embryos, 1:1 diploid-aneuploid mosaic embryos show developmental potential equivalent to diploids. Their peri-implantation development was followed, and it was found that while aneuploid cells in the EPI underwent apoptosis, the diploid cells over-proliferated to regulate the overall EPI size. These results elucidate the cellular and molecular mechanisms used by mouse embryo to refine the EPI cell population and ensure only the chromosomally fit cells proceed through the development of the fetus. The second part of the study investigates into the early molecular players that bias cell fate decisions. Sox21 was earlier identified as the most heterogeneous gene at the 4-cell stage that can influence cell fate decision. The deep sequencing of Sox21 knockout and wild-type embryos was carried out at the 4-cell stage and compared. Klf2 and Tdgf1 were found to be important downstream targets of Sox21 that influence lineage segregation. Depletion of both these genes predisposed cells to the TE lineage. Co-overexpression of both these genes rescued the effect of Sox21 knockdown on cell fate. These results demonstrate the mechanism by which Sox21 heterogeneity, from as early as the 4-cell stage, biases cell fate. Together, these findings indicate the fundamental mechanisms used by mouse embryo to ensure developmental plasticity

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs