scholarly journals Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres

Reproduction ◽  
2005 ◽  
Vol 129 (5) ◽  
pp. 547-556 ◽  
Author(s):  
Sigrid Eckardt ◽  
N Adrian Leu ◽  
Satoshi Kurosaka ◽  
K John McLaughlin
Keyword(s):  
Somatic Cell ◽  
Fluorescent Protein ◽  
Cumulus Cell ◽  
Cell Nuclei ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Somatic Genome ◽  
Cleavage Stages ◽  
Green Fluorescent

Mammalian somatic cell cloning requires factors specific to the oocyte for reprogramming to succeed. This does not exclude that reprogramming continues during the zygote and cleavage stages. The capacity or role of zygotic and cleavage stages to reprogram somatic cell nuclei is difficult to assess due to the limited development of somatic cell nuclei transplanted into cytoplasts of these stages. Alternatively, tetraploid embryos have been used to study reprogramming and can be assessed for their contribution to extra-embryonic lineages. When mouse cumulus cell nuclei transgenic for Oct4-green fluorescent protein (GFP) were injected into intact two- and four-cell stage blastomeres, manipulated embryos developed into blastocysts with expression of Oct4-GFP as observed in embryos produced by nuclear transfer into metaphase II oocytes. However, only the latter contributed to extra-embryonic tissues in day 10.5 conceptuses, with the exclusion of the somatic genome in cells originating from transfer into blastomeres already at 5.5 days post conception. Somatic nuclei transferred into cleavage stage blastomeres reinitiated expression of an embyronic-specific transgene, but lacked the extent of reprogramming required for contribution to postimplantation development, even when complemented by an embryonic genome.

43 PATTERN OF NUCLEOLI FORMATION IN RACCOON-PORCINE INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 169
Author(s):  
Y. H. Nam ◽  
Y. Jeon ◽  
S. A. Cheong ◽  
S. S. Kwak ◽  
S. H. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Species ◽  
Housekeeping Genes ◽  
Blastocyst Stage ◽  
Control Group ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Embryonic Genome

Recently, great focus has been on the rescue of endangered animals through somatic cell nuclear transfer (SCNT). Because it is difficult to obtain the oocytes of endangered species, interspecies SCNT (iSCNT) methods have been attempted. Numerous iSCNT embryos have shown unsuccessful development due to aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). In particular, aberrant EGA may cause the arrest of nucleoli formation and developmental block in embryos. According to this concept, we performed raccoon iSCNT using porcine oocytes and analyzed iSCNT embryo development pattern and formation of nucleoli. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. Cleavage and blastocyst formation were evaluated under a stereomicroscope at 48 and 168 h post-activation (hpa), respectively. To confirm the formation of nucleoli, which can be detected by C23 antibody labeling in many mammalian species, C23 immunocytochemistry was performed at 48 and 72 hpa. A total of 158 iSCNT embryos were cultured; 68.5% of the raccoon iSCNT embryos were cleaved at 48 hpa (1-cell stage: 9.7%; 2-cell stage: 14.4%; 4-cell stage: 34.1%; 6-cell stage: 12.7%; 8-cell stage: 7.3%; fragmented: 21.8%). But, the embryos seen as 5- to 8-cell stage did not have the same number of nuclei as their blastomere number. When raccoon iSCNT embryos were stained by Hoechst 33342, 5- to 8-blastomere raccoon iSCNT embryos had only 4 nuclei. The raccoon iSCNT embryos did not develop past the 4-cell stage and failed to form blastocysts. In the control group, 65.2% of pig SCNT embryos were cleaved at 48 hpa (1-cell stage: 8.0%; 2-cell stage: 4.2%; 4-cell stage: 23.6%; 6-cell stage: 13.6%; 8-cell stage: 23.8%; fragmented: 26.8%), and 10.0% of pig SCNT embryos developed to blastocysts. In raccoon iSCNT embryos, raccoon nuclei failed to form nucleoli at 48 and 72 hpa. By contrast, pig SCNT embryos showed 18.8 and 87.9% nucleoli formation at 48 and 72 hpa. Our results demonstrate that 4-cell-stage embryos of raccoon-porcine hybrid embryos may be produced by SCNT methods. The pig oocytes partly supported the remodeling and reprogramming of the raccoon somatic cell nuclei, but they were unable to support nucleoli formation. Moreover, aberrant nucleoli formation caused the unsuccessful development of raccoon SCNT embryos to the blastocyst stage. This work was supported by a grant from the Next Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Incorporation of Uridine in Cleavage Stage Eggs of the Sea Urchin Paracentrotus Lividus

1971 ◽  
Vol 26 (8) ◽  
pp. 816-821 ◽  
Author(s):  
Larry E. Bockstahler
Keyword(s):  
Ion Exchange ◽  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Paracentrotus Lividus ◽  
Cleavage Stage ◽  
Early Cleavage ◽  
Cell Stage ◽  
Cleavage Stages ◽  
Layer Chromatography

Incorporation of uridine in cleavage stage eggs of the sea urchin Paracentrotus lividus was investigated. It was shown by ion exchange and thin layer chromatography that most of the uridine taken up during the 16-cell stage was converted into UTP with some incorporation into UDP and UMP. Conversion of uridine to these phosphorylated nucleosides occurred throughout early cleavage stages. A very small amount of uridine taken up by cleavage stage eggs is incorporated into RNA heterogeneous in size. This RNA was examined by polyacrylamide gel electrophoresis.

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.

Expression of Green Fluorescent Protein in Canine-Cloned Embryos Following Interspecies Somatic Cell Nuclear Transfer.

2008 ◽  
Vol 78 (Suppl_1) ◽  
pp. 92-92
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Min Kyu Kim ◽  
Hyun Ju Oh ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Green Fluorescent

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.

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.

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