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.

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 Effect of microinjection of CRISPR / Cas9 components in plasmid form on the development of rabbit embryos during in vitro culture

2020 ◽  
Author(s):  
E.M. Koloskova ◽  
◽  
V.A. Ezerskii ◽  
T.P. Trubitsina ◽  
◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Housekeeping Genes ◽  
Cell Stage ◽  
Embryo Survival ◽  
Genetic Construct ◽  
Stage Of Development ◽  
High Concentrations ◽  
Green Fluorescent ◽  
Rabbit Embryos

The survival rate of rabbit embryos microinjected by the plasmid form of CRISPR/Cas9 components specific to the sour whey protein gene was evaluated. At high concentrations of plasmid components, embryo survival decreased slightly, possibly because the WAP gene does not belong to the housekeeping genes. After microinjection of a genetic construct with a sequence of green fluorescent protein under a cytomegalovirus promoter, the embryo survival significantly decreased. This is most likely due to the superexpression of GFP at the 2-16 cell stage of development.

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.

21 SITE-SPECIFIC RECOMBINATION USING Dre-RECOMBINASE IN PORCINE CELLS AND EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
S. Y. Yum ◽  
S. J. Kim ◽  
J. H. Moon ◽  
W. J. Choi ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cellular Level ◽  
Cell Stage ◽  
Site Specific ◽  
Target Gene Expression ◽  
Green Fluorescent ◽  
Gateway Cloning System

Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.

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.

scholarly journals Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

Reproduction ◽  
2009 ◽  
Vol 138 (5) ◽  
pp. 783-791 ◽  
Author(s):  
Tereza Toralová ◽  
Andrej Šušor ◽  
Lucie Němcová ◽  
Kateřina Kepková ◽  
Jiří Kaňka
Keyword(s):  
Fluorescent Protein ◽  
Staining Intensity ◽  
Preimplantation Development ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Average Decrease ◽  
Complex Protein ◽  
Green Fluorescent

Identification of genes that are important for normal preimplantation development is essential for understanding the basics of early mammalian embryogenesis. In our previous study, we have shown that CENPF (mitosin) is differentially expressed during preimplantation development of bovine embryos. CENPF is a centromere–kinetochore complex protein that plays a crucial role in the cell division of somatic cells. To our best knowledge, no study has yet been done on either bovine model, or oocytes and preimplantation embryos. In this study, we focused on the fate of bovine embryos after injection of CENPF double-stranded RNA (dsRNA) into the zygotes. An average decrease of CENPF mRNA abundance by 94.9% or more and an extensive decline in immunofluorescence staining intensity was detected relative to controls. There was no disparity between individual groups in the developmental competence before the 8-cell stage. However, the developmental competence rapidly decreased then and only 28.1% of CENPF dsRNA injected 8-cell embryos were able to develop further (uninjected control: 71.8%; green fluorescent protein dsRNA injected control: 72.0%). In conclusion, these results show that depletion of CENPF mRNA in preimplantation bovine embryos leads to dramatic decrease of developmental competence after embryonic genome activation.

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.

scholarly journals Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development

Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3467-3474 ◽  
Author(s):  
M.A. Ciemerych ◽  
D. Mesnard ◽  
M. Zernicka-Goetz
Keyword(s):  
Fluorescent Protein ◽  
Polar Body ◽  
Lineage Tracing ◽  
Embryonic Axes ◽  
Cell Stage ◽  
Adult Mice ◽  
Vegetal Pole ◽  
Mammalian Embryos ◽  
Pole Cells ◽  
Green Fluorescent

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591–5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.

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.

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