Abstract:
:The potential applications of a simplified method of somatic cell nuclear transfer (SCNT) that is improved in both efficiency and throughput is considerable. Technically, a major step of SCNT is to produce large pools of enucleated oocytes (cytoplasts) efficiently, a process that requires considerable micromanipulation skill and expensive equipment. Here, we have developed an efficient and high-throughput method of manual oocyte enucleation using a simple device, a pulled Pasteur pipette, that can be connected to standard zona-free method of embryo reconstitution. Common Pasteur pipettes were pulled on a flame to produce finely drawn pipettes with inner diameters approximately less than half the oocyte diameter (∼50-60 μm), and slightly larger than cytoplasmic protrusion (∼20-30 μm) that was induced after demecolcine treatment of MII-stage oocytes. Oocyte manipulation was performed under a stereomicroscope by either bisecting the oocyte into two approximately equal demioocytes (blind manual enucleation), or by positioning the oocytes so that the cytoplasmic extrusion that contains the MII chromosome mass is removed with the minimum amount of cytoplasm (oriented manual enucleation). The survival rate of the manually enucleated oocytes was 81.4-91.5%, comparable to standard zona-free method of oocyte enucleation (>95%). A total of 80-120 oocytes could be enucleated in 10 min, which was considerably higher than standard zona-free enucleation method. In vitro development rates of cloned embryos derived from manually enucleated cytoplasts with varying cytoplasmic volumes (50%, 95%, and 100%) was comparable, and embryonic developmental rates of the two latter groups were at least as good as standard zona-free method. The manual method of oocyte enucleation described here can be learned and mastered for simple, fast, and cheap production of cloned embryos with comparable efficiency to other available methods.
journal_name
Cell Reprogramjournal_title
Cellular reprogrammingauthors
Hosseini SM,Hajian M,Moulavi F,Asgari V,Forouzanfar M,Nasr-Esfahani MHdoi
10.1089/cell.2012.0033subject
Has Abstractpub_date
2013-02-01 00:00:00pages
15-23issue
1eissn
2152-4971issn
2152-4998journal_volume
15pub_type
杂志文章abstract::Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2012.0052
更新日期:2013-04-01 00:00:00
abstract::Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0005
更新日期:2014-08-01 00:00:00
abstract::Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2015.0065
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abstract::The purpose of this study was to investigate the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on transgene expression and development of porcine transgenic cloned embryos, specifically focusing on effects derived from TSA-treated donor cells or TSA-treated reconstructed embryos. The results...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0041
更新日期:2015-02-01 00:00:00
abstract::The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression o...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0036
更新日期:2014-12-01 00:00:00
abstract::Based on microarray data comparing gene expression of fibroblast donor cells and bovine somatic cell nuclear transfer (SCNT) and in vivo produced (AI) blastocysts, a group of genes including several transcription factors was selected for evaluation of transcript abundance. Using SYBR green-based real-time polymerase c...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2009.0042
更新日期:2010-02-01 00:00:00
abstract::The generation of induced pluripotent stem cells (iPSCs) from somatic cells by expressing ectopic reprogramming transcriptional factors such as Oct3/4, Sox2, Klf4, c-Myc, and Nanog is one of the cutting-edge discoveries in stem cell and cancer research. This discovery has raised several safety issues regarding the use...
journal_title:Cellular reprogramming
pub_type: 杂志文章,评审
doi:10.1089/cell.2013.0077
更新日期:2014-04-01 00:00:00
abstract::Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with f...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0072
更新日期:2015-06-01 00:00:00
abstract::As man's best friend, dogs have an important position in human society. Ten years ago, we reported the first cloned dog, and his birth has raised various scientific issues, such as those related to health, reproduction, and life span. He has developed without any unique health issues. In this article, we summarize and...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2015.0033
更新日期:2015-10-01 00:00:00
abstract::Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experim...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2011.0012
更新日期:2011-10-01 00:00:00
abstract::Induced pluripotent stem cells (iPSCs) have an extensive application in regenerative medicine, pharmaceutical discovery, and basic research. With the recent derivation of rat iPSCs, it is now feasible to apply genetic manipulation in this species. But such tools do not yet exist for many rat strains, especially for di...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2016.0028
更新日期:2017-06-01 00:00:00
abstract::The scope of this article is not to provide an exhaustive review of nuclear transfer research, because many authoritative reviews exist on the biological issues related to somatic and embryonic cell nuclear transfer. We shall instead provide an overview on the work done specifically on sheep and the value of this work...
journal_title:Cellular reprogramming
pub_type: 杂志文章,评审
doi:10.1089/cell.2013.0032
更新日期:2013-10-01 00:00:00
abstract::The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as w...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2009.0049
更新日期:2010-04-01 00:00:00
abstract::Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs an...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2009.0093
更新日期:2010-08-01 00:00:00
abstract::Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2015.0009
更新日期:2015-10-01 00:00:00
abstract::Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that c...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2010.0022
更新日期:2011-02-01 00:00:00
abstract::Epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndMT) describe complex changes in progenitor lineage, cell morphology, and gene expression. Stimulated by environmental cues, these cellular transitions are essential for elements of embryonic development and can be pathologically d...
journal_title:Cellular reprogramming
pub_type: 杂志文章,评审
doi:10.1089/cell.2012.0054
更新日期:2013-04-01 00:00:00
abstract::The generation of defined somatic cell types from pluripotent stem cells represents a promising system for many applications for regenerative therapy or developmental studies. Certain key developmental genes have been shown to be able to influence the fate determination of differentiating stem cells suggesting an alte...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2011.0067
更新日期:2012-04-01 00:00:00
abstract::Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scient...
journal_title:Cellular reprogramming
pub_type: 传,历史文章,杂志文章
doi:10.1089/cell.2013.0038
更新日期:2013-10-01 00:00:00
abstract::It has been demonstrated that several types of somatic stem cells have the remarkable capacity to differentiate into other types of tissues. However, the promise of keratinocyte stem cells seems slim for generating nonepidermal tissues. Using our recently developed acclimatization induction strategy, we demonstrate th...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2009.0095
更新日期:2010-06-01 00:00:00
abstract::Buffalo embryos were produced by hand-made cloning using skin fibroblasts from male and female buffaloes (n = 4 each) as donor cells for examining the effect of sex. Although the rate of blastocyst formation (43.8% ± 1.31% vs. 42.2% ± 1.22%) was similar, the total cell number (333 ± 10.4 vs. 270 ± 10.9) was higher (p ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2015.0077
更新日期:2016-10-01 00:00:00
abstract::Induced pluripotent stem cells (iPSCs) play an important role in cell replacement therapy. Several studies have shown that keratinocytes are promising reprogrammed cells. We easily and efficiently enriched epidermal stem cells by attaching them for a limited time in culture dishes. Individual epidermal cells enriched ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2018.0035
更新日期:2018-12-01 00:00:00
abstract::Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such a...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0028
更新日期:2014-10-01 00:00:00
abstract::Kunming (KM) mice are the most widely used strain in China. However, authentic embryonic stem cells (ESCs) from KM mice have never been available, and this hampers the genetic manipulation of this valuable mice strain. In this study, we show that KM ESCs can be efficiently derived and maintained in chemically defined ...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2012.0065
更新日期:2013-06-01 00:00:00
abstract::In this study, we found that the measles virus (MV) can infect human-induced pluripotent stem cells (hiPSCs). Wild-type MV strains generally use human signaling lymphocyte activation molecule (SLAM; CD150) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both CD150 and CD46 as recepto...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2017.0034
更新日期:2018-02-01 00:00:00
abstract::A simple, rapid, efficient, and specialized culture system was successfully developed in this study to induce human embryonic stem cells into dopaminergic neurons in vitro. It only took 5 days to generate quickly and directly a large number of homogeneous neural stem cell (NSC) spheres by the introduction of small mol...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2019.0037
更新日期:2019-12-01 00:00:00
abstract::The differentiation of multipotent stem cells toward a pancreatic lineage provides us with an alternative cell-based therapeutic approach to type 1 diabetes and enables us to study pancreas development. The current study aims to study the effect of growth factors such as activin A or nicotinamide, alone and in combina...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2016.0043
更新日期:2017-08-01 00:00:00
abstract::Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic a...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2010.0047
更新日期:2011-04-01 00:00:00
abstract::In contrast to adult stem cells, induced pluripotent stem cells (iPSCs) can be grown robustly in vitro and differentiated into virtually any tissue, thus providing an attractive alternative for biomedical applications. Although iPSC technology is already being used in human biomedicine, its potential in animal product...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2014.0087
更新日期:2015-06-01 00:00:00
abstract::Edaravone can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells and replace lost cells by transplanting neuron-like cells to repair spinal cord injury (SCI). In this study, BMSCs were derived from the bone marrow of male Wistar rats (4 weeks old) through density gradient centr...
journal_title:Cellular reprogramming
pub_type: 杂志文章
doi:10.1089/cell.2020.0055
更新日期:2021-01-05 00:00:00