scholarly journals Genome stabilization by RAD51‐stimulatory compound 1 enhances efficiency of somatic cell nuclear transfer‐mediated reprogramming and full‐term development of cloned mouse embryos

2021 ◽  
Author(s):  
Ah Reum Lee ◽  
Ji‐Hoon Park ◽  
Sung Han Shim ◽  
Kwonho Hong ◽  
Hyeonwoo La ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Full Term ◽  
Genome Stabilization

Kdm6a overexpression improves the development of cloned mouse embryos

Zygote ◽  
2017 ◽  
Vol 26 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Guang-yu Bai ◽  
Si-hang Song ◽  
Yu-wei Zhang ◽  
Xiang Huang ◽  
Xing-wei Huang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Life Science ◽  
Cumulus Cell ◽  
Science Research ◽  
Mouse Embryos ◽  
Rdna Transcription ◽  
X Chromosomes ◽  
Blastocyst Rate

SummarySomatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

31 FULL-TERM AND LIVE RABBIT CLONES PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 124 ◽  
Author(s):  
F. Du ◽  
J. Xu ◽  
S. Gao ◽  
L. Y. Sung ◽  
D. Stone ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Fusion ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Full Term ◽  
Fusion Method ◽  
Nuclear Injection

Transgenic/knockout (KO) rabbits can serve as an excellent animal model for human cardiovascular diseases (CVD) and other diseases. However, the production of transgenic/KO rabbits is hindered by low efficiency of traditional DNA microinjection and the unavailability of embryonic stem cell lines. An alternative approach is to produce transgenic/KO rabbits by somatic cell nuclear transfer (SCNT) using genetically modified somatic cells as nuclear donors. Our initial objective of the study was to prove the feasibility of cloning rabbits by SCNT because rabbit is a difficult species to be cloned. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of follicle-stimulating hormone (FSH) and human choriani gonadotropin (hCG). Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in M199 + 10% fetal bovine serum (FBS) and confirmed by fluorescence microscopy. Cumulus cells used for nuclear donors were prepared from fresh cumulus-oocytes complexes. The donor nucleus was transferred into a recipient oocyte by either cell fusion or direct nuclear injection method. In the cell fusion method, a small donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte; subsequently the somatic cell-cytoplast pair was fused by applying three direct current pulses at 3.2 kV/cm for a duration of 20 µs/pulse. In the direct nuclear injection method, a mechanically lysed donor cell was injected into oocyte cytoplasm with the aid of a piezo-drill system. Fused embryos or injected oocytes were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg/mL cycloheximide for 2 h. For the in vitro study, cloned embryos were cultured in B2 medium plus 2.5% FBS for 5 days (initiation of activation = day 0) at 38.5°C in 5% CO2 humidified air. For the in vivo study, cloned embryos were cultured for 20–22 h in vitro before transfer into pseudopregnant rabbit recipients. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post embryo transfer (ET). The results (Table 1) show that the donor nuclei-introducing rate was higher with nuclear direct injection than with the cell fusion method (P < 0.05). There were no significant differences among subsequent cleavage and development to morula and blastocysts between both methods, although the development rates of cloned embryos via electrically mediated fusion were higher than those derived from the injection group. One recipient in the injection group (1/6, 17%) and six recipients in the fusion group (6/16, 38%) were diagnosed as pregnant. From the fusion group, one full-term but stillborn and one live and healthy clone rabbit were delivered on Days 33 and 31 post-ET, respectively. To our knowledge, this is the second report of full term development of cloned rabbit by somatic nuclear transfer cloning. Our further study is to clone live rabbit offspring with modified transgenic/KO somatic cell lines. Table 1. In vitro development of rabbit cloned embryos with cumulus cells as nuclear donors This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261–11.

Full-Term Development of Gaur–Bovine Interspecies Somatic Cell Nuclear Transfer Embryos: Effect of Trichostatin A Treatment

2012 ◽  
Vol 14 (3) ◽  
pp. 248-257 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Sumeth Imsoonthornruksa ◽  
Chuti Laowtammathron ◽  
Anawat Sangmalee ◽  
Wanchai Tunwattana ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Full Term

Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer

2020 ◽  
Author(s):  
Taiki Shindo ◽  
Shunya Ihashi ◽  
Yuko Sakamoto ◽  
Tomomi Okuno ◽  
Junko Tomikawa ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Actin Filaments ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Nuclear Actin ◽  
Actin Assembly ◽  
Filamentous Form ◽  
Nuclear Events ◽  
Dynamic Assembly

Abstract Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.

Abnormal dynamic changes in β-tubulin in somatic nuclear transfer cloned mouse embryos

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 76-82 ◽  
Author(s):  
Jingling Shen ◽  
Zhendong Wang ◽  
Xinghui Shen ◽  
Zhong Zheng ◽  
Qinghua Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitotic Division ◽  
Mouse Embryos ◽  
Dynamic Changes ◽  
Microtubule Network ◽  
Multipolar Spindles ◽  
Β Tubulin ◽  
Cellular Organelles

SummaryThe efficiency of somatic cell nuclear transfer (SCNT) cloning remains low, thus limiting the applications of this technique. In this study, we used immunochemistry and confocal microscopy to detect the microtubule component, β-tubulin, in SCNT, parthenogenetic (PA), and intracytoplasmic sperm injection (ICSI) embryos before the first mitotic division. β-Tubulin is the component subunit of microtubule, which plays critical roles in regulating localization of cellular organelles, and the growth, maturation and fertilization of oocytes. Our results demonstrated similar changes of spindle patterns in PA and ICSI embryos. The second meiotic division resumed 1 h post-treatment, and the cytoplasmic asters (CAs) disappeared. After about 4–6 h of treatment, pronuclei formed with the midbodies connecting each other. Meanwhile, the CAs reappeared and a microtubule network developed in the cytoplasm. However, SCNT embryos showed abnormal multipolar spindles, and the pseudopronuclei that contained many nucleoli existed after 6 h of SrCl2 activation. Enucleated oocytes alone did not form spindle-like structures when they were artificially activated for 6 h, indicating that somatic cell chromosomes might be necessary for spindle formation in SCNT embryos. These results demonstrated abnormal changes of β-tubulin in mouse SCNT embryos, compared with PA and ICSI embryos.

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