402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
M. K. Gupta ◽  
S. J. Uhm ◽  
E. Y. Kim ◽  
Y. H. Jung ◽  
J. Y. Yu ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Standard Procedure ◽  
Metaphase Plate ◽  
Egfp Expression ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Transgenic Livestock ◽  
Expression Rate

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng �L-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

301 EVALUATION OF THE SPERM-MEDIATED GENE TRANSFER (SMGT) TECHNIQUE BY IN VITRO FERTILIZATION IN PIGS USING RecA PROTEIN

2008 ◽  
Vol 20 (1) ◽  
pp. 230
Author(s):  
F. A. García-Vázquez ◽  
A. Gutiérrez-Adán ◽  
J. Gadea
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Reca Protein ◽  
Control Group ◽  
Cleavage Rate ◽  
Competitive System ◽  
Experimental Conditions ◽  
Exogenous Dna ◽  
Vitro Fertilization

Sperm-mediated gene transfer (SMGT) is a transgenesis technique used in pigs mainly byAI (Lavitrano ML et al. 2002 Proc. Natl. Acad. Sci. USA 99, 14 230–14 235), and by intracytoplasmic spermi injection (ICSI) (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338), but up to now, it has not been reported by IVF (Bolling LC et al. 2003 Transgenics 4, 77–86). The aim of this study was to evaluate the efficiency of SMGT by IVF in pigs and the use of recombinase RecA to avoid possible exogenous DNA degradation by endonucleases. We designed a study with 3 experimental groups: (1) sperm incubated without exogenous DNA (control group), (2) sperm incubated with exogenous DNA (DNA group), and (3) sperm incubated with the complex RecA:DNA (RecA group). Ejaculates from mature boars were recovered and the seminal plasma was discarded to avoid detrimental effects on DNA binding. The spermatozoa were incubated with DNA or RecA-DNA and used as vectors for transferring linealized plasmid DNA [5.7 kb enhanced green fluorescent protein (EGFP)] into in vitro-matured porcine oocytes by IVF. Spermatozoa and oocytes were coincubated for 2 h in TALP medium; then, the fertilized oocytes were transferred into the culture drops with NCSU-23 medium. The binding of the DNA to the spermatozoa was confirmed by the use of enzymatic fluorescein-12-dUTP-labeled DNA and measured by flow cytometry. The total number of oocytes used was 584 (n = 59; n = 382; n = 143 for the 3 experimental groups, respectively). Embryos were examined for cleavage rate at 48 h after fertilization, and for embryo development at 144 h. Expression of EGFP in embryos was examined using a fluorescence inverted microscope. The results in our experiment showed that the coincubation of sperm with exogenous DNA induced a lower cleavage rate than when the RecA:DNA complex was used (DNA: 25.13 � 2.22 v. RecA: 41.26 � 4.13%, P < 0.05), and both no different from the control group (38.98 � 6.40). On the other hand, the production of blastocysts was similar in the 3 groups (Control: 21.74 � 8.79 v. DNA: 21.87 � 4.24 v. RecA: 15.25 � 4.72%) as well as the quality of the obtained embryos. The average number of cells per blastocyst was similar in the 3 groups (36.40 � 9.28 v. 37.26 � 3.32 v. 28.45 � 3.34, respectively). None of the produced embryos was detected for expressing protein EGFP. In conclusion, under our experimental conditions, IVF is not an effective technique for SMGT, whereas using ICSI-SMGT under the same conditions (DNA and DNA:RecA groups), we obtained a high percentage of transgenic embryos (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). Three main causes are hypothesized to be probably related to this conclusion: (i) the penetration of the oocytes is achieved only by the not DNA-bound viable spermatozoa in a competitive system, (ii) the DNA was only bound to altered membrane or dead spermatozoa, and (iii) the exogenous DNA is only present on sperm surface and in the process of fusion with oocyte membrane, the DNA is not internalized.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577 ◽  
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Abstract Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

84 PAIRS OF BLASTOMERES FROM BOVINE DAY 5 MORULAE ARE ABLE TO CONTRIBUTE TO INNER CELL MASS AND TROPHECTODERM IN CHIMERIC EMBRYOS GENERATED BY AGGREGATION WITH TWO DAY 4 MORULAE

2015 ◽  
Vol 27 (1) ◽  
pp. 135
Author(s):  
K. Simmet ◽  
M. Reichenbach ◽  
S. Jung ◽  
R. Fries ◽  
T. Grupp ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Standard Procedure ◽  
Cell Mass ◽  
Breeding Value ◽  
Egfp Expression ◽  
Promising Alternative ◽  
Donor Embryo

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 339-346 ◽  
Author(s):  
Liangxue Lai ◽  
Qingyuan Sun ◽  
Guangming Wu ◽  
Clifton N. Murphy ◽  
Birgit Kühholzer ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Pronuclear Formation

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8±17.3% vs 28.5±3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0±7.0% vs 63.3±12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0±11.6% vs 4.6±4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

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.

248 IN VITRO PRODUCTION OF CATTLE × BUFFALO HYBRID EMBRYOS USING BOVINE OOCYTES AND AFRICAN BUFFALO (SYNCERUS CAFFER CAFFER) EPIDIDYMAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 240
Author(s):  
O. D. Owiny ◽  
D. M. Barry ◽  
M. Agaba ◽  
R. A. Godke
Keyword(s):  
Bison Bison ◽  
Abnormal Development ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Epididymal Sperm ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Interspecies hybridization of bovids occurs between domestic cattle and at least 3 other species: the American bison (Bison bison), yak (Bos grunniens), and banteng (Bos banteng). Birth of a cattle � buffalo hybrid was reported in Russia, but the report was never authenticated. Such hybrids could be important in improving livestock production and managing diseases that impede production in tropical Africa. We investigated hybridization between cattle and their closest African wild relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce pre-implantation cattle � buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to a standard IVF procedure with either homologous (n = 1166 oocytes) or heterologous (n = 1202 oocytes) buffalo epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate. Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of cleaved homologous embryos progressed to the morula stage compared with 12.7% for hybrids. No hybrid embryos developed beyond the 16-cell stage, whereas 40.1% of the cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization, and absence of nuclei in some blastomeres were common in the hybrid embryos. We conclude that interspecies fertilization of cattle oocytes with African buffalo sperm occurs in vitro and that the barrier to hybridization is in the early stages of embryonic development. Also, chromosomal disparity is the likely cause of fertilization abnormalities, abnormal development, and subsequent arrest, impairing the formation of pre-implantation hybrid embryos. Investigation into developmental abnormalities, including reciprocal hybridization and genetic studies of the hybrid embryos, are recommended.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

310 PRODUCTION OF TRANSGENIC CLONED PORCINE EMBRYOS EXPRESSING EGFP OR LacZ GENE THROUGH REPLICATION DEFECTIVE RETROVIRAL VECTOR

2008 ◽  
Vol 20 (1) ◽  
pp. 235
Author(s):  
S. J. Uhm ◽  
M. K. Gupta ◽  
T. Kim ◽  
H. T. Lee
Keyword(s):  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Fluorescein Isothiocyanate ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Lacz Gene ◽  
Cell Stage ◽  
Uv Illumination ◽  
Green Fluorescent

We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 � 6.5, 53.6 � 6.4, and 12.0 � 5.7% for EGFP; 83.5 � 8.2, 57.5 � 6.3 and 10.1 � 4.1% for LacZ; and 80.5 � 4.2, 60.9 � 8.2 and 12.3 � 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos. This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.

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