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.

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)

scholarly journals 175 ESTABLISHMENT OF PORCINE EMBRYONIC STEM CELL LINE DERIVED FROM IN VIVO BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 238 ◽  
Author(s):  
S.-A. Ock ◽  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
L.-Y. Shi ◽  
S.-L. Lee ◽  
...  
Keyword(s):  
Cell Line ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Feeder Layer ◽  
Es Cell ◽  
Stem Cell Line

A porcine embryonic stem (ES) cell line was established from an in vivo-flushed blastocyst. The present study evaluated the effectiveness of IVP, parthenotes and in vivo-produced embryos on establishment of an ES cell line. IVP blastocysts were produced from slaughterhouse ovaries based on the previously reported protocols (2000 Theriogenology 54, 787–797) with minor modifications. Parthenote blastocysts were produced by activation of oocytes matured in vitro with electric stimulation of 2 DC pulses at 2.0 kV/cm for 30 μsec in 0.3 M mannitol solution containing 100 μM CaCl2 and 100 μM MgCl2 in vivo blastocysts were recovered on Day 7 after AI (Day = 0) by flushing the uterus with D-PBS containing 10% FBS from three females. After removal of zona pellucida with 0.2% pronase, the blastocysts were subjected to immunosurgical treatment with 10% rabbit anti-pig serum to isolate the inner cell mass (ICM) as previously reported (1975 PNAS 72, 5099–5102). The ICM was seeded onto the feeder layer of STO which was inactivated by treatment with 10 μg/mL mitomycin for 2.5 h and cultured in DMEM with 0.1 mM β-mercaptoethanol, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 0.1 mM MEM non-essential amino-acid, 20 ng/mL rh-bFGF, 40 ng/mL rh-LIF, 0.03 mM adenosine, 0.03 mM guanosine, 0.03 mM cytidine, 0.03 mM uridine, 0.01 mM thymidine, and 15% FBS. The culture was maintained by changing the medium every day after initiation of ICM attachment onto the feeder layer. Any ES-like colonies were individually picked off the feed layer, dissected with 0.25% trypsin-0.02% EDTA for 3–5 min and reseeded on to new STO feed layer. Out of 140 blastocysts (25, in vivo; 55, IVF; 60, parthenotes) used, attaching rates of the ICMs onto the feeder layer were 88% (22/25, in vivo), 56.4% (31/55, IVF), and 58.3% (35/60, parthenotes). A total of 15 primary ES-like colonies was formed in in vivo (3, 12%), IVF (5, 9.1%), and parthenote (7, 11.7%). However, only one ES cell line from in vivo blastocyst was established, which was confirmed as positive by AP activity (Promega, Madison, WI, USA), and was maintained through four passages. In conclusion, for establishment of an ES cell line in pig, the in vivo blastocyst method is superior to currently available methods utilizing IVF or parthenotes. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

197 PRODUCTION OF IN VITRO- AND IN VIVO-DERIVED CAT BLASTOCYSTS FOR THE ESTABLISHMENT OF FELINE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 207 ◽  
Author(s):  
J. Kehler ◽  
M. Roelke-Parker ◽  
B. Pukazhenthi ◽  
W. Swanson ◽  
C. Ware ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chorionic Gonadotropin ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Feeder Layers

Identification and characterization of spontaneously occurring genetic diseases in cats has permitted the development of valuable models for testing potential treatments of similar human diseases. With the near completion of the feline genome project, establishment of pluripotential feline embryonic stem (ES) cells would facilitate the targeting of specific genetic loci to produce new feline medical models. Two approaches were used to produce feline blastocysts in an attempt to establish feline ES cells in culture. Naive queens were superovulated with an intramuscular (i.m.) injection of 150 IU of equine chorionic gonadotropin (eCG) followed by an i.m. injection of 100 IU of human chorionic gonadotropin (hCG) 80 h later; follicles were aspirated laparoscopically 24-26 h later for subsequent in vitro fertilization (IVF). On average, 29 mature cumulus oocyte cell complexes (COCs) were recovered from each queen. IVF was performed in 50 microliter drops of complete Hams F-10 medium containing 30 000 fresh, motile sperm. COCs were cultured overnight in 5% carbon dioxide at 38�C, and residual adherent cumulus cells were removed 12 to 16 h later by trituration in 0.1% hyaluronidase. Embryos were cultured in fresh drops of Hams F-10, and on average 25% developed to the early blastocyst stage after 7 days. Alternatively, estrus was induced in queens with a single i.m. injection of 100 IU of eCG, and then 72 h later queens were permitted six supervised matings with a fertile tom over the next two days. Queens underwent ovariohysterectomy 7 days after their first copulation, and compacted morulae and early blastocysts were flushed from the oviducts and uterine horns. On average, eight embryos were recovered from the reproductive tract of each queen. Both in vivo- and in vitro-matured blastocysts were subsequently cultured in standard mouse ES cell medium on inactivated mouse embryonic fibroblasts. When they failed to hatch in culture after 3 days, a 0.5% pronase solution was used to dissolve the zonae pellucidae under microscopic visualization. Denuded expanded blastocysts adhered to the heterotypic feeder layer and primary inner cell mass (ICM) outgrowths formed within 4 days. Outgrowths were mechanically disaggregated into small clusters of 15 to 20 cells and re-plated on fresh feeders. These colonies grew slowly and were transferred after one week onto new feeder layers. The addition of murine or human recombinant leukemia inhibitory factor had no effect on the survival and proliferation of primary outgrowths or subsequent colonies. After 3 weeks, all colonies derived from both in vivo- and in vitro-matured blastocysts had either differentiated or died. Additional experiments are ongoing to test the effects of homotypic feeder layers and alternative growth factors on promoting the establishment and survival of feline ES cell lines. Ultimately, germline transmission of any putative feline ES cell lines will need to be demonstrated in vivo for their utility in gene targeting experiments to be realized.

281 ATTEMPTS FOR ESTABLISHMENT OF PORCINE EMBRYONIC STEM-LIKE CELLS DERIVED FROM IN VITRO-PRODUCED BLASTOCYSTS

2009 ◽  
Vol 21 (1) ◽  
pp. 237
Author(s):  
H. M. Kim ◽  
J. K. Park ◽  
S. G. Lee ◽  
C. H. Park ◽  
S. W. Yoon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Control Group ◽  
Es Cell ◽  
Cell Stage

The porcine embryonic stem (ES) cells could be a useful tool for the production of transgenic animals and the study of developmental gene regulation. Even though the efficiency of establishment of ES cells from in vivo blastocysts is relatively high, especially in mice, it is difficult and expensive to obtain in vivo embryos in domestic animals. Recent development of techniques in the production of embryos in vitro could be a useful source for the establishment of ES cells. However, the morphology and cell quality of in vitro-produced embryos are inferior to those of their in vivo counterparts. Although many attempts have been made to establish ES cells from in vitro-produced embryos, the overall efficiency is extremely low because of the poor embryo quality. However, aggregation of in vitro-produced embryos was developed to increase the number of cells in the inner cell mass (ICM) of blastocysts and could be useful in the application to ES cell establishment. Therefore, in this study, we attempted to derive porcine ES cells by using aggregation of in vitro-produced embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Embryos at the 4-cell stage were produced by culturing embryos for 2 days after IVF and SCNT. After removal of the zona pellucida with acid Tyrode’s solution, three 4-cell-stage embryos (IVF3X) from IVF and two 4-cell-stage embryos (NT2X) from SCNT were aggregated by co-culturing them in an aggregation plate followed by culturing to the blastocyst stage. Embryos from IVF (IVF control) and SCNT (NT control) were also cultured to the blastocyst stage. All blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Two primary colonies were formed in the IVF control group (3.9%), whereas four primary colonies were formed in the IVF3X group (12.5%). One primary colony was formed in the NT2X group (20%), although no colony was formed in the NT control group. One of the IVF3X lines gradually disappeared after sub-passing, and the NT2X line also disappeared. Two ES-like cell lines derived from the IVF control were maintained up to 14 passages, and three ES-like lines from IVF3X were also maintained for more than 14 passages. These cells morphologically resembled human ES cells (flat and single layered) and expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, Oct-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. These results indicated that a porcine ES cell line could be established from in vitro-produced aggregated blastocysts. Further research is required to establish ES cell lines from SCNT embryos and characterize the differentiation and developmental abilities of these porcine ES-like cells. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

scholarly journals In vivo culture of bovine embryos and quality assessment of in vivo vs. in vitro produced embryos

2012 ◽  
Vol 50 (No. 4) ◽  
pp. 149-158 ◽  
Author(s):  
V. Havlicek ◽  
M. Lopatarova ◽  
S. Cech ◽  
R. Dolezel ◽  
T. Huber ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Recovery Rate ◽  
Bovine Embryos ◽  
In Vivo Culture ◽  
Blastocyst Rate ◽  
Culture Procedure ◽  
Recovery Methods

Routine access to the bovine oviduct for in vivo culture accomplishes various demands on embryo production for scientific as well as commercial purposes. The experiments conducted in the present study focused on the efficiency of recovery methods after temporary in vivo culture of bovine embryos in oviducts of the homologous species using transvaginal endoscopy (Experiment I) and on the quality assessment of recovered blastocysts (Experiment II). In Experiment I in vitro matured oocytes were fertilized, cultured for 1 to 3 days and transferred unilaterally into the ipsilateral oviducts of 54 heifers by the means of transvaginal endoscopy. After 4 to 6 days of in vivo culture embryos were re-collected either by non-surgical flushing of uterine horns (U-group) or by combined flushing of the oviducts and uterine horns (OU-group). In total the recovery rate was 38.4% (780/2029). After flushing at day seven, 106 blastocysts (blastocyst rate: 13.6% ) were found. The additional 24 h of in vitro culture (day eight) resulted in 153 blastocysts (blastocyst rate: 19.6% ). The recovery rate in the OU-group was twice as efficient as in the U-group (390/1358 vs. 390/671, P < 0.01). The recovery rates among the different stages of transferred embryos did not differ significantly; likewise cross-effects among the stages and the recovery methods were non-significant. The recovery methods (P < 0.001) and the interaction between the recovery methods and the stages of transferred embryos (P < 0.01) had an influence on blastocyst yields on day seven (U-group 37/1358 vs. OU-group 69/671) and day eight (U-group 48/1358 vs. OU-group 105/671). In Experiment II embryo quality was assessed by the survival rate of blastocysts after freezing in ethylene glycol. Day seven embryos were produced in vitro (in vitro group D7) or by IVM/IVF followed by a combined culture procedure (2 to 3 days in vitro prior to 4 to 5 days in vivo) (in vivo group D7) or after superovulation and collection at day seven (superovulation group). Embryos from in vitro group D7 re-expanded only for 6 h after thawing, embryos from in vivo group D7 and superovulation group were alive for 24 h and 72 h of culture, respectively. Only embryos derived by superovulation showed hatching activity. Blastocysts from the in vitro group D7 and the in vivo group D7 that were held in culture medium for additional 24 h (day eight) showed an analogous post-thawing culture behaviour. In conclusion, the results of the present study demonstrated that some embryos transferred for in vivo culture remain in the oviduct even at day seven. Hence, combined flushing of oviducts and uterine horns after in vivo culture in the bovine oviduct is necessary for effective embryo re-collection. The quality of recovered embryos after temporary in vivo culture assessed by cryotolerance was in-between those produced in vitro or recovered after superovulation.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
D. M. de Souza ◽  
H. Fernandes ◽  
P. V. Silva ◽  
B. Cazari ◽  
P. D. Moço ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Training Model ◽  
Perivitelline Space ◽  
Chi Square ◽  
Testicular Cells ◽  
Chi Square Test ◽  
Cellular Components

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.

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