41 UPDATING THE ZONA-FREE METHOD FOR MOUSE CLONING USING HM1 EMBRYONIC STEM CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
I. Lagutina ◽  
M. Lizier ◽  
M. Paulis ◽  
F. Lucchini ◽  
A. Castelli ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Farm Animals ◽  
Blastocyst Development ◽  
Donor Cells ◽  
M Phase ◽  
Fetal Bovine ◽  
Spherical Cells ◽  
Old Mice

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.Partially supported by grant Superpig from Regione Lombardia.

scholarly journals 173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 237 ◽  
Author(s):  
G. Lazzari ◽  
I. Lagutina ◽  
G. Crotti ◽  
P. Turini ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Es Cell ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

334 FACTORS AFFECTING HEMATOPOIETIC ENGRAFTMENT OF MONKEY EMBRYONIC STEM CELLS IN SHEEP FETUSES

2015 ◽  
Vol 27 (1) ◽  
pp. 255
Author(s):  
Y. Nagao ◽  
T. Abe ◽  
A. Hara ◽  
B. Sarentonglaga ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Subcutaneous Tissue ◽  
Es Cells ◽  
Embryonic Stem ◽  
Factors Affecting ◽  
Colony Pcr ◽  
Donor Cells ◽  
Haematopoietic Tissue ◽  
Cell Layers ◽  
Differentiation Stage ◽  
Teratoma Formation

Previously, we generated monkey/sheep haematopoietic chimeras by in utero transplantation (IUT) of monkey embryonic stem (ES); however, the factors that control how the ES cells successfully engraft and differentiate into haematopoietic tissue in sheep fetuses remain uncertain. Here, we examined factors that might influence donor cells and recipient sheep and affect successful ES cell engraftment. We transplanted either undifferentiated monkey ES cells or ES-derived cells at an early haematopoietic differentiation stage into sheep fetuses. The latter cells were allowed to differentiate by culturing on OP9 cell layers for 6 days. Cells were transplanted into the liver or subcutaneous tissue of recipient sheep fetuses at 43 to 50 or 51 to 67 days of gestation (full term = 147 days) using ultrasound to identify the site for transplantation. After birth, monkey haematopoietic engraftment in the bone marrow was analysed in 40 lambs using colony-PCR with cells grown in methylcellulose in the presence of defined cytokines; teratoma formation was analysed by biopsy and immunohistochemistry. We found that haematopoietic engraftment was only observed when ES-derived cells at the early differentiation stage were transplanted into fetal livers at 51 to 67 days of gestation (6/9). However, teratoma formation with mature monkey tissue structures was only observed following transplantation of undifferentiated ES cells into fetal subcutaneous tissues at 43 to 50 days of gestation (4/6), but that was not observed when both types of cells were transplanted into the liver (0/18) or at 51 to 67 days of gestation (0/24). These results demonstrate that the differentiation status of the donor cells, the transplantation site, and the age of the fetus at transplantation are important factors in engraftment and differentiation into haematopoietic tissue or teratoma formation in sheep fetuses.

56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

2008 ◽  
Vol 20 (1) ◽  
pp. 109 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
X. L. Jin ◽  
Y. Y. Lee ◽  
Y. J. Cho ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cumulus Cells ◽  
Complete Medium ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Frozen Semen ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL–1 hCG, and 10 ng mL–1 epidermal growth factor (EGF) at 38.5�C and 5% CO2 in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 � 106 sperm mL–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean � SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 � 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 � 2.7%, 38/45), and iSCNT (89.3 � 2.7%, 100/110, monkey; 89.3 � 3.3%, 45/49, pig; and 86.0 � 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 � 3.0%, 2/49, and 7.9 � 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 � 2.9%, 38/117, and 26.7 � 2.8%, 12/88, respectively; P < 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 � 2.7%, 78.1 � 2.1%, and 82.3 � 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.

294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT

2011 ◽  
Vol 23 (1) ◽  
pp. 244
Author(s):  
R. Dutta ◽  
D. Malakar ◽  
K. Khate ◽  
J. Akshay
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Patient Specific ◽  
Fibroblast Cells ◽  
Donor Cells ◽  
Adult Fibroblasts

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance. The authors acknowledge N.D.R.I for the financial and infrastructural assistance.

scholarly journals Puerarin Exerts a Delayed Inhibitory Effect on the Proliferation of Cardiomyocytes Derived from Murine ES Cells via Slowing Progression through G2/M Phase

2016 ◽  
Vol 38 (4) ◽  
pp. 1333-1342 ◽  
Author(s):  
Xueying Luo ◽  
Binlong Zhong ◽  
Xian Hong ◽  
Yurong Cui ◽  
Ying Gao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Early Stage ◽  
Es Cells ◽  
Embryonic Stem ◽  
Specific Inhibitor ◽  
Cyclin B1 ◽  
Inhibitory Effect ◽  
Cell Cycle Distribution ◽  
M Phase ◽  
Cyclin A2

Objective: Puerarin, which shows beneficial and protective effects on cardiovascular diseases, is the main isoflavone extracted from Pueraria lobata (kudzu) root. The aim of this study was to investigate the effects of puerarin on in vitro myocardial proliferation and its underlying mechanism. Methods: Myocardial differentiation of transgenic embryonic stem (ES) cells was performed by embryoid body-based differentiation method. The proliferation assay of cardiomyocytes (CMs) derived from ES cells (ES-CMs) was performed by EdU (5-Ethynyl-2'-deoxyuridine) staining. Flow cytometry was employed to determine the cell cycle distribution and apoptosis of purified ES-CMs. Quantitative real-time PCR was utilized to study the transcription of genes related to cell cycle progression. Signaling pathways relating to proliferation were studied by western blot analysis and application of specific inhibitors. Results: Puerarin exerted a delayed inhibitory effect on the proliferation of ES-CMs at the early-stage differentiation. Meanwhile, puerarin slowed progression through G2/M phase without inducing apoptosis of ES-CMs. Further assays showed that puerarin up-regulated the transcription of Cyclin A2, Cyclin B1 and Cdk1 in ES-CMs. The ERK1/2 specific inhibitor PD0325901 and the PI3K specific inhibitor Wortmannin successfully reversed puerarin-induced up-regulation of Cdk1 but not Cyclin A2 and B1. Conclusion: These findings suggest that puerarin inhibits CM proliferation via slowing progression through G2/M phase during early-stage differentiation.

Strategies for the isolation and characterization of bovine embryonic stem cells

1994 ◽  
Vol 6 (5) ◽  
pp. 569 ◽  
Author(s):  
RA Cherny ◽  
TM Stokes ◽  
J Merei ◽  
L Lom ◽  
MR Brandon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Isolation And Characterization

The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and culture of pluripotent cells from in vitro produced porcine embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Ming Li ◽  
Yong-Hai Li ◽  
Yi Hou ◽  
Xiao-Fang Sun ◽  
Qingyuan Sun ◽  
...  
Keyword(s):  
Fetal Bovine Serum ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Embryonic Fibroblasts ◽  
Feeder Layers ◽  
Edta Solution ◽  
Fetal Bovine

The present study was designed to examine whether in vitro produced porcine embryos can be used to establish an embryonic stem (ES) cell line. Porcine embryos were produced by in vitro maturation and in vitro fertilization. Embryos at the 4-cell to blastocyst stages were cultured in an ES medium containing 16% fetal bovine serum with mouse embryonic fibroblasts as a feeder layer. It was found that ES-like colonies were derived only from blastocysts. When these ES-like colonies were separated in 0.25% trypsin–0.02% EDTA solution and cultured again, ES-like colonies were further observed in the subsequent culture until the fourth passage. The cells from ES-like colonies showed positive alkaline phosphatase activity. Some cells from the colonies differentiated into several types of cells in vitro when they were cultured in the medium without feeder layers and leukemin inhibitory factor. Embryoid bodies were also formed when the cells were cultured in a suspension status. These results indicate that porcine ES-like cells can be derived from in vitro produced porcine blastocysts and these ES-like cells are pluripotent. The culture system used in the present study is useful to isolate and culture ES cells from in vitro produced porcine embryos.

scholarly journals 61 CLONED MOUSE PRODUCED USING A ZONA FREE METHOD OF NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 180
Author(s):  
R. Ribas ◽  
B. Oback ◽  
J. Taylor ◽  
A. Maurício ◽  
M. Sousa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Strontium Chloride ◽  
Embryonic Cells ◽  
Reconstructed Embryos

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.

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