Biomedical subjects

X Cindy Tian

Publications and source records attributed to X Cindy Tian.

At least 19 recordsLinked to original sources

PubMed2007

Risk assessment of meat and milk from cloned animals.

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.

Animals↗

PubMed2006

Premature chromosome condensation is not essential for nuclear reprogramming in bovine somatic cell nuclear transfer.

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%-91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

Animals↗

PubMed2006

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Adult Stem Cells↗

PubMed2006

XIST repression in the absence of DNMT1 and DNMT3B.

X chromosome inactivation (XCI) in human and mice involves XIST/Xist gene expression from the inactive X (Xi) and repression from the active X (Xa). Repression of the XIST/Xist gene on the Xa has been associated with methylation of its 5' region. In mice, Dnmt1 has been shown to be involved in the methylation and transcriptional repression of Xist on Xa. We examined maintenance of XIST gene repression on Xa in HCT116 cell lines knockout for either DNMT1 or DNMT3B and for DNMT1 and DNMT3B simultaneously. Methylation of the XIST promoter and XIST transcriptional repression is sustained in DNMT1-, DNMT3B- and DNMT1/DNMT3B knockout cells. Despite global DNA demethylation, the double knockout cells present only partial demethylation of the XIST promoter, which is not sufficient for gene reactivation. In contrast, global DNA demethylation with 5-aza-2'-deoxycytidine leads to XIST expression. Therefore, in these human cells maintenance of XIST methylation is controlled differently than global genomic methylation and in the absence of both DNMT1 and DNMT3B.

Cell Line↗

PubMed2006

Bovine embryonic stem cells.

Bovine embryonic stem (bES) cell lines reported to date vary in morphology and marker expression, such as alkaline phosphatase (ALPL), stage-specific embryonic antigen 4 (SSEA4), and octamer-binding transcription factor-4 (OCT4), that normally are associated with the undifferentiated, pluripotent state. This chapter introduces the methods of isolating and maintaining bovine ES cells. These bovine ES cells grow in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells, as well as human EG cells. Throughout the culture period, most of the cells within the colonies stain positive for ALPL and cell surface markers SSEA4 and OCT4. The staining patterns of the bES cells are identical to those of the blastocysts fertilized in vitro (IVF), yet different from most previously reported bovine ES cell lines, which are either negative or not detected. After undifferentiated culture for more than 1 year, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. In addition to bES from IVF, this chapter introduces two methods of generating blastocyst stage embryos other than in vitro fertilization, which are parthenogenetic activation and somatic cell nuclear transfer for the potential application of generation "patient-specific" ES cells.

Animals↗

PubMed2006

Cardiomyocytes.

Derivation of cardiomyocytes from embryonic stem cells would be a boon for treatment of the many millions of people worldwide who suffer significant cardiac tissue damage in a myocardial infarction. Such cells could be used for transplantation, either as loose cells, as organized pieces of cardiac tissue, or even as pieces of organs. Eventual derivation of human embryonic stem cells via somatic cell nuclear cloning would provide cells that not only may replace damaged cardiac tissue, but also would replace tissue without fear that the patient's immune system will reject the implant. Embryonic stem cells can differentiate spontaneously into cardiomyocytes. In vitro differentiation of embryonic stem cells normally requires an initial aggregation step to form structures called embryoid bodies that differentiate into a wide variety of specialized cell types, including cardiomyocytes. This chapter discusses methods of encouraging embryoid body formation, causing pluripotent stem cells to develop into cardiomyocytes, and expanding the numbers of cardiomyocytes so that the cells may achieve functionality in transplantation, all in the mouse model system. Such methods may be adaptable and/or modifiable to produce cardiomyocytes from human embryonic stem cells.

Animals↗

PubMed2006

Engineering cardiac tissue from embryonic stem cells.

Restoration of cardiac function by replacement of diseased myocardium with functional cardiac myocytes may offer a potential cure for cardiac disease and will likely revolutionize treatment methods. During the past 20 years, we have seen the development of tissue engineering; among these types of tissue engineering is cardiac tissue engineering. This type of cardiac tissue engineering includes growing neonatal cardiomyocytes on preformed polymers, liquid collagen, and temperature-responsive surfaces. It also includes the application of neonatal rat or chick cardiomyocytes to skeletal myoblasts, mesenchymal stem cells and embryonic stem cells, static culture, and bioreactor and stretching cultivation. Progress has come step-by-step, but, in recent years, with great technological advances, the progress has been accelerating, moving this area of research from dream to reality. The engineered cardiac tissue not only reproduces in vitro, but it can also be shaped so that it will, at some time, be able to form valves or endothelial lining. This chapter describes the currently used protocols for cardiac tissue engineering: liquid collagen-based cardiac tissue engineering and cell sheet-based cardiac tissue engineering, especially cardiac tissue engineering using cardiomyocytes derived from embryonic stem cells.

Animals↗

PubMed2006

Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes.

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

Anaphase↗

PubMed2005

Global gene expression profiles reveal significant nuclear reprogramming by the blastocyst stage after cloning.

Nuclear transfer (NT) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. Here, we compared global gene expression profiles of individual bovine NT blastocysts with their somatic donor cells and fertilized control embryos using cDNA microarray technology. The NT embryos' gene expression profiles were drastically different from those of their donor cells and closely resembled those of the naturally fertilized embryos. Our findings demonstrate that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, problems may occur during redifferentiation for tissue genesis and organogenesis, and small reprogramming errors may be magnified downstream in development.

Animals↗

PubMed2005

Promoter-specific expression of the imprinted IGF2 gene in cattle (Bos taurus).

The domestic cattle (Bos taurus) has been a good animal model for embryo biotechnologies, such as in vitro fertilization and nuclear transfer. However, animals produced from these technologies often suffer from large-calf syndrome, suggesting fetal growth disregulation. The product of the insulin-like growth factor 2 (IGF2) gene is one of the most important fetal mitogens known to date. A detailed analysis of age-, tissue-, and allele-specific expression of IGF2 has not been performed in the bovine mainly because the majority of the bovine sequence has been unavailable. In the present study, we obtained virtually the entire sequence of the bovine IGF2 cDNA, identified expressed single-nucleotide polymorphisms (SNPs) in both exons 3 and 10, and determined the age-, tissue-, and promoter-specific expression of bovine IGF2 in fetal, calf, and adult tissues. We found that, similar to the human and mouse, bovine IGF2 is subjected to extensive transcriptional regulation through multiple promoters, alternative splicing and polyadenylation, as well as genetic imprinting. However, major differences were found in the regulation of the bovine IGF2 in nearly all aspects of age-, tissue-, promoter-, and allele-specific expression of IGF2, and the promoter-specific loss of imprinting from every other species studied, including cattle's close relatives, the sheep and the pig. The data presented here are of important reference value to cattle produced from embryo biotechnologies.

Age Factors↗

PubMed2005

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

Animals↗

PubMed2005

Meat and milk compositions of bovine clones.

The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones.

Animals↗

PubMed2005

Generation and characterization of pluripotent stem cells from cloned bovine embryos.

Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nuclear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-staining patterns identical to those of the bovine blastocysts.

Alkaline Phosphatase↗

PubMed2005

A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos.

MacroH2A histones are variants of canonical histone H2A that are conserved among vertebrates. Previous studies have implicated macroH2As in epigenetic gene-silencing events including X chromosome inactivation. Here we show that macroH2A is present in developing and mature mouse oocytes. MacroH2A is localized to chromatin of germinal vesicles (GV) in both late growth stage (lg-GV) and fully grown (fg-GV) stage oocytes. In addition, macroH2A is associated with the chromosomes of mature oocytes, and abundant macroH2A is present in the first polar body. However, maternal macroH2A is lost from zygotes generated by normal fertilization by the late 2 pronuclei (2PN) stage. Normal embryos at 2-, 4-, and 8-cell stages lack macroH2A except in residual polar bodies. MacroH2A protein expression reappears in embryos after the 8-cell stage and persists in morulae and blastocysts, where nuclear macroH2A is present in both the trophectodermal and inner cell mass cells. We followed the loss of macroH2A from pronuclei in parthenogenetic embryos generated by oocyte activation. Abundant macroH2A is present upon the metaphase II plate and persists through parthenogenetic anaphase, but macroH2A is progressively lost during pronuclear decondensation prior to synkaryogamy. Examination of embryos generated by intracytoplasmic sperm injection (ICSI) revealed that macroH2A is associated exclusively with female pronuclei prior to loss in late pronucleus stage embryos. These results outline a surprising finding that a maternal store of macroH2A is removed from the maternal genome prior to synkaryogamy, resulting in embryos that execute three to four mitotic divisions in the absence of macroH2A prior to the onset of embryonic macroH2A expression.

Animals↗

PubMed2005

Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones.

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Animals↗

PubMed2004

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification.

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.

Animals↗

PubMed2004

Genomic imprinting of H19 in naturally reproduced and cloned cattle.

Animals produced from assisted reproductive technologies suffer from developmental abnormalities and early fetal death at a higher frequency than that observed in those produced by natural breeding. These symptoms are reminiscent of imprinting disruptions in the human and mouse, suggesting the possibility of perturbations in the expression of imprinted genes such as biallelic expression or silencing. H19 is one of the imprinted genes first identified in mice and humans, but its sequence and imprinting status have not been determined in cattle. In the present study, we obtained the majority of the bovine H19 gene sequence (approximately 2311 base pairs), identified a single nucleotide polymorphism (SNP) in exon 5 and determined the frequencies of different alleles containing the SNP. Our analysis demonstrated that, in cattle produced by natural breeding, H19 was indeed imprinted as shown by either predominant or exclusive expression of the maternal allele. We also analyzed the imprinting pattern of H19 in organs of four animals produced by somatic cell nuclear transfer that died shortly after birth or had developed abnormalities that necessitated immediate killing at birth. Three out of four cloned animals showed biallelic expression of H19, supporting our hypothesis that imprinting disruption is present in cloned animals that suffered from developmental abnormalities at birth. Examination of the expression of H19 in the offspring of a cloned animal produced by artificial insemination showed that the imprinting pattern in this animal was indistinguishable from those of control animals, suggesting that either imprinting disruptions in cloned animals are corrected through natural reproduction or that they are not present in healthy cloned animals capable of undergoing natural reproduction.

Alleles↗

PubMed2004

Serial bull cloning by somatic cell nuclear transfer.

Although the list of species successfully cloned continues to grow, serial cloning has not been reported in species other than the mouse. Here we describe two live births of second-generation clones of a bull. Clones of the first and second generations appear healthy and have normal telomere lengths. Our attempts to produce the third generation of clones were unsuccessful.

Abortion, Veterinary↗