Recloned transgenic pigs possess normal reproductive performance and stable genetic transmission capacity

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Zubing Cao ◽  
Yan Li ◽  
Xiao Wen ◽  
Zhiyuan Li ◽  
Changsheng Mi ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Fluorescent Protein ◽  
Sperm Concentration ◽  
Full Term ◽  
Transmission Capacity ◽  
Transgenic Pigs ◽  
Genetic Transmission ◽  
Sperm Volume ◽  
Transgenic Embryos ◽  
Green Fluorescent

SummaryThe present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

scholarly journals Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

2014 ◽  
Vol 60 (3) ◽  
pp. 230-237 ◽  
Author(s):  
Hitomi MATSUNARI ◽  
Toshihiro KOBAYASHI ◽  
Masahito WATANABE ◽  
Kazuhiro UMEYAMA ◽  
Kazuaki NAKANO ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transgenic Pigs ◽  
Specific Expression ◽  
Green Fluorescent

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 < 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P < 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

Production of transgenic rabbit embryos through intracytoplasmic sperm injection

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Qiuyan Li ◽  
Jian Hou ◽  
Sheng Wang ◽  
Yongfu Chen ◽  
Xiao-Rong An
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Fluorescent Protein ◽  
Dna Uptake ◽  
Dna Encoding ◽  
Cell Stage ◽  
Transgenic Rabbit ◽  
Dna Attachment ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Rabbit Embryos

SummaryThe objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4–9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/μl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

scholarly journals GATA Motifs Regulate Early Hematopoietic Lineage-Specific Expression of the Gata2 Gene

2005 ◽  
Vol 25 (16) ◽  
pp. 7005-7020 ◽  
Author(s):  
Maki Kobayashi-Osaki ◽  
Osamu Ohneda ◽  
Norio Suzuki ◽  
Naoko Minegishi ◽  
Tomomasa Yokomizo ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Domain ◽  
Gata Factor ◽  
Specific Expression ◽  
Developmental Potential ◽  
Gfp Reporter ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Definitive Hematopoiesis

ABSTRACT Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5′ to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP+ fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34+/c-Kit+ cells than the GFP− fraction did. When cultured in vitro, the P-Sp GFP+ cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp.

Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies

2011 ◽  
Vol 20 (9) ◽  
pp. 1563-1575 ◽  
Author(s):  
Monika Nowak-Imialek ◽  
Wilfried A. Kues ◽  
Bjoern Petersen ◽  
Andrea Lucas-Hahn ◽  
Doris Herrmann ◽  
...  
Keyword(s):  
Animal Model ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Large Animal Model ◽  
Large Animal ◽  
Transgenic Pigs ◽  
Green Fluorescent

128 MULTIPLICATION OF 8-CELL EMBRYOS BY AGGREGATION OF A SINGLE ENHANCED GREEN FLUORESCENT PROTEIN-LABELED BLASTOMERE WITH PUTATIVE TETRAPLOID EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 168
Author(s):  
M. I. Hiriart ◽  
R. J. Bevacqua ◽  
R. Fernandez-Martin ◽  
D. F. Salamone
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Egfp Expression ◽  
Simple Method ◽  
Transgenic Embryos ◽  
Green Fluorescent

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.

Ultrastructure of vitrified rabbit transgenic embryos

Zygote ◽  
2013 ◽  
Vol 22 (4) ◽  
pp. 558-564 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich ◽  
M. Popelková ◽  
J. Schlarmannová ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Ultrastructural Level ◽  
Membrane Structures ◽  
Egfp Gene ◽  
Cell Structures ◽  
Morula Stage ◽  
Transgenic Embryos ◽  
Green Fluorescent

SummaryThe aim of the study was to determine the viability of rabbit transgenic (enhanced green fluorescent protein (EGFP)-positive) embryos cultured in vitro and compare with gene-microinjected (Mi) non-transgenic (EGFP-negative) embryos following vitrification. Non-microinjected and non-vitrified embryos were used as the control. Morphological signs of injury to embryo organelles were determined at the ultrastructural level using transmission electron microscopy (TEM). Morphometric evaluation was performed on cellular organelles using microphotographs obtained by TEM. Intact and Mi embryos recovered from in vivo fertilized eggs at 19–20 hours post coitum (hpc) were cultured for up to 72 hpc (morula stage), evaluated for the EGFP gene integration and then vitrified in 0.25 ml insemination straws in modified EFS (40% ethylene glycol + 18% Ficoll 70 + 0.3 M sucrose) vitrification solution. After 1–3 days the embryos were devitrified, a representative selection of embryos was analyzed by TEM and the remaining embryos were subjected to additional in vitro culture. Observations by TEM showed that the vitrified/warmed EGFP-positive and EGFP-negative embryos had a slight accumulation of cellular debris and lipid droplets compared with the control intact embryos. More severe changes were detected in the membrane structures of the treated embryos, mostly in the cytoplasmic envelope, trophoblastic microvilli, junctional contacts and mitochondria. We suggest that the higher proportion of deteriorated cell structures and organelles in the treated embryos may be due to the vitrification process rather than to mechanical violation (the gene-microinjection procedure), as a detailed inspection of ultrastructure revealed that most damage occurred in the cell membrane structures.

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