56 TRANSGENESIS BY HANDMADE CLONING USING EGFP-TRANSFECTED YUCATAN FIBROBLASTS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
P. M. Kragh ◽  
Y. Du ◽  
J. Li ◽  
Y. Zhang ◽  
L. Bolund ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Cumulus Cells ◽  
Miniature Pig ◽  
Egfp Gene ◽  
Fusion Procedure ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, genetic manipulations can be performed in cells isolated from special breeds followed by SCNT into enucleated oocytes isolated from slaughterhouse ovaries. In the present study, we established production of Yucatan blastocysts by the handmade cloning (HMC) technique using non-transgenic fibroblasts from the Yucatan miniature pig, and produced transgenic blastocysts using enhanced green fluorescent protein (EGFP)-positive Yucatan fetal fibroblasts. For transgenesis, Yucatan fibroblasts from a 40-day old fetus were transfected with a vector containing an EGFP gene and a neomycin-resistance selection gene by lipofection. Well separated neomycin-resistant colonies were isolated, expanded, and used for HMC. For HMC, cumulus–oocyte complexes were aspirated from ovaries of slaughterhouse sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The remaining parts, i.e. cytoplasts, were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused with one fibroblast in the absence of Ca2+. After one h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, and subsequently cultured in cytochalasin B and cycloheximide for 4 h. The development of reconstructed embryos to the blastocyst stage was determined after 7 days of in vitro culture. When using non-transgenic and EGFP-positive Yucatan fetal fibroblasts, the rate of blastocyst formation (mean � SEM) were 36 � 7% (36/102) and 42 � 7% (32/77), respectively. In conclusion, the HMC technique was very efficient for production of blastocysts of Yucatan miniature pig origin using both non-transgenic and EGFP-positive fetal fibroblasts.

34 PIGLETS BORN FROM HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 135 ◽  
Author(s):  
Y. Du ◽  
Y. Zhang ◽  
J. Li ◽  
P. M. Kragh ◽  
M. Schmidt ◽  
...  
Keyword(s):  
New Technology ◽  
Calf Serum ◽  
Cumulative Effect ◽  
Cumulus Cells ◽  
Widespread Application ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) is probably the most efficient way to produce pigs with targeted genetic modification. Handmade cloning (HMC) is a new technology for SCNT developed recently (Vajta et al. 2005 Reprod. Fertil. Dev. 17, 97–112). However, HMC that resulted in births in cattle was regarded technically difficult in pigs due predominantly to the fragility of MII phase porcine oocytes. The purpose of our present work was to use optimized porcine HMC for production of cloned piglets. Data were analyzed by t-test using SPSS (11.0; SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. Oriented handmade enucleation was performed as described elsewhere (Li et al. concomitant abstract). Briefly, oocytes that were partially digested with 3.3 mg mL-1 of pronase for 20 s were removed of polar bodies (PB) and adjacent small volume of cytoplasm by manual bisection in HEPES-buffered TCM-199 medium supplemented with 2% calf serum and 2.5 �g mL-1 of cytochalasin B. Halves without PB were collected as putative cytoplasts. In vitro-cultured porcine fetal fibroblasts were used as donor cells. After cytoplast-fibroblast pairing, fusion and activation of fused cytoplast-fibroblast pairs (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Clon Stem Cells 7, 199–205), reconstructed embryos were cultured in a modified well of the well (WOW) culture system (Feltrin et al. 2006 Reprod. Fertil. Dev. 18, 126 abst) with porcine zygote medium-3 (PZM3) supplemented with 4 mg mL-1 of BSA. The cumulative effect of the optimization steps has resulted in considerably improved in vitro efficiency, shown as 64 � 2.3 (mean � SEM) reconstructed embryos from 151.3 � 4.8 oocytes could be obtained per day after 3–4 h manual work, including a 1-h pause between fusion and activation. One-half (50.1 � 2.8%) of the reconstructed embryos developed to the blastocyst stage after 7 days, a rate that was significantly higher than that obtained with traditional cloning (TC; 27.7 � 2.2%; P < 0.01). To compare the transfer efficiency between HMC and TC, blastocysts from both HMC and TC produced by using nuclear donor cells of different origin, respectively, to identify the offspring were transferred surgically to synchronized recipients. Of 6 pregnancies produced, 2 are ongoing, 2 were lost, and 2 term litters of 3 and 10 piglets were born. Live birth/transferred embryo efficiencies for HMC and TC are 17% (10/58) and 15% (3/20). According to our knowledge, a litter size of 10 cloned healthy piglets, as achieved in this study, is the highest one that ever has been reported. Our data suggest that porcine HMC is a very promising method for SCNT and may promote its widespread application for various purposes.

scholarly journals 75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD FOR HANDMADE CLONING IN CATTLE AND SWINE

2004 ◽  
Vol 16 (2) ◽  
pp. 159 ◽  
Author(s):  
G. Vajta ◽  
T.T. Peura ◽  
L. Lai ◽  
C.N. Murphy ◽  
R.S. Prather ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Cumulus Cells ◽  
Dependent Manner ◽  
Uv Illumination ◽  
Reconstructed Embryo ◽  
Fetal Fibroblasts ◽  
Membrane Protrusions ◽  
Handmade Cloning

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P<0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 148 ◽  
Author(s):  
J. Li ◽  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
...  
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
Uv Illumination ◽  
Polar Bodies ◽  
Transgenic Embryos ◽  
Handmade Cloning

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P &lt; 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P &lt; 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells. Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 169
Author(s):  
H. M. Zhou ◽  
Y. Chen
Keyword(s):  
Skin Fibroblast ◽  
Polar Body ◽  
Subsequent Development ◽  
Fibroblast Cells ◽  
Fetal Skin ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
First Polar Body ◽  
Fetal Fibroblasts

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P &gt; 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

scholarly journals 28 SIMPLIFIED ACTIVATION METHOD TO IMPROVE THE IN VITRO DEVELOPMENT OF HANDMADE CLONED (HMC) PORCINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 114
Author(s):  
Y. Du ◽  
Z. Yang ◽  
B. Lv ◽  
L. Lin ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Cell Number ◽  
Activation Method ◽  
The Other ◽  
Electrical Activation ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Fetal Fibroblasts ◽  
Group 2 ◽  
Vitro Development

Delayed activation is commonly used in pig somatic cell nuclear transfer (SCNT) where electrical activation is followed by chemical activation. However, chemical incubation of several hours (up to 4 or 6) is logistically not very convenient even though handmade cloning (HMC) could improve the overall efficiency of pig cloning (Du et al. 2007 Theriogenology 68, 1104–1110). It was reported that a brief exposure of cycloheximide (CX) before electrical activation could significantly increase developmental rate and total blastocyst cell number when simultaneous activation was performed in micromanipulator-based pig cloning (Naruse et al. 2007 Theriogenology 68, 709–716). The purpose of our present work is to investigate whether such activation method is also applicable for pig HMC. Data were analyzed by t-test using SPSS (11.0, SPSS Inc., Chicago, IL, USA). After 42 h in vitro maturation, cumulus cells were removed. In vitro-cultured porcine fetal fibroblasts were used as donor cells. Cytoplast-fibroblast pairing, electrical fusion and activation of fused cytoplast-fibroblast pairs were performed as described previously (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205). Three groups were compared due to different activation protocol. In Group 1 (control), reconstructed embryos were cultured in porcine zygote medium 3 (PZM3) supplemented with 4 mg mL–1 BSA, 5 μg mL–1 cytochalasin B (CB), and 10 μg mL–1 CX for 4 h. In Group 2 (CX priming), fused pairs and the other halves of cytoplasts were incubated in HEPES-buffered TCM-199 medium supplemented with 10% calf serum, 10 μg mL–1 CX for 10 min just before the second fusion or electrical activation. In Group 3 (CB + CX priming), treatment similar to Group 2 was performed except that additional 5 μg mL–1 CB was added for the 10-min incubation. Reconstructed embryos were in vitro cultured in the well of the well (WOW) system for 6 days. Blastocyst rates and total cell numbers of Day 6 blastocysts were evaluated. As illustrated in Table 1, embryos pretreated with both CB and CX gave the best results, with better blastocyst formation (53.8 ± 4.8%; mean ± SEM) and higher cell number (77.2 ± 5.4) compared to the other 2 groups. Our data suggested that CX and CB priming could be used as a solution to the long chemical incubation in porcine SCNT by HMC, making the embryos more receptive to electrical activation. Table 1.In vitro development of HMC reconstructed embryos with different activation protocols

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

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