• Title/Summary/Keyword: Tetraploidy

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Tetraploidy Induction of Mouse Embryos by In Vitro Culture with Cytochalasin B (Cytochalasin B를 이용한 체외배양에 의한 생쥐 수정란의 4배체 유도)

  • 진동일
    • Journal of Embryo Transfer
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    • v.14 no.2
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    • pp.89-92
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    • 1999
  • 효율적인 homozygous 동물을 생산하기 위한 실험의 단계로 염색체가 4배체인 수정란의 이용성을 타진하기 위해 생쥐 수정란과 cytochalasin B를 사용하여 4배체 유도에 관한실험을 수행하였다. 생쥐 2-세포기 수정란을 10$\mu\textrm{g}$/ml 농도의 cytochasin B로 약 20시간 배양하였을 때 모든 수정란은 발육을 거의 멈추었으나, 이 수정란을 cytochalasin B-free medium에 체외배양하였을 때 발육이 재개되어 48시간 후 상실기나 배반포기까지 약 74%의 발육율을 나타내었다. 그러나 발육된 수정란의 세포수는 대조구에 비해 휠신 적은 것으로 나타났다. 염색체 분석결과 cytochalasin B로 처리한 대부분의 수정란은 4배체인 것으로 나타났고 약간의 수정란은 mosaicism과 다배체를 나타내기도 하였다. 그러므로 cytochalasin B를 이용하여 효과적으로 4배체의 수정란을 유도할 수 있는 것으로 나타났다.

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Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells

  • Shin, Dong-Hyuk;Lee, Jeoung-Eun;Eum, Jin Hee;Chung, Young Gie;Lee, Hoon Taek;Lee, Dong Ryul
    • Development and Reproduction
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    • v.21 no.4
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    • pp.425-434
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    • 2017
  • Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

Morphological and Cytogenetic Analysis of Colchicine-induced Tetraploids of Fallopia multiflolra Haraldson (Colchicine 처리에 의해 유기된 4배체 하수오의 형태 및 세포유전학적 특성)

  • Kim, Ki Hyun;Youn, Cheol Ku;Kim, In Jae;Lee, Kyung Ja;Kim, Young Ho;Hong, Seong Tack;Woo, Sun Hee
    • Korean Journal of Medicinal Crop Science
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    • v.26 no.5
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    • pp.362-369
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    • 2018
  • Background: For stable induction of tetraploidy in Fallopia multiflora Haraldson, colchicine was treated to establish the condition of induction and investigated the morphological and cytogenetic traits of the tetraploid plants obtained compared to those of diploid ones. Methods and Results: For the induction of tetraploidy, F. multiflora plants were soaked in aqueous solutions of colchicine at various concentration (0.1, 0.5, and 1.0%). After this, 2% dimethyl sulfoxide (DMSO) was added at room temperature on a shaker set at 150 rpm for periods of 12, 24, and 48 h. The induction rate of tetraploids appeared to be the highest in plants treated with 0.5% colchicine for 24 h. As the colchicine concentration and soaking time increased above these levels, the growing tip of the roots did not develop and they began to rot. When compared to diploid plants, tetraploids differed greatly in various characteristics, including the sizes and shapes of the leaves, fruits, flowers and roots. The induced tetraploid F. multiflora had larger guard cells, and chloroplasts, increased number of chloroplast in the guard cells and decreased stomatal densities. Conclusions: When colchicine induced plants for tetraploid, it can be distinguished from diploids, in various characteristics such as morphological changes as stomatal size, number of chloroplasts per guard cell, number of chromosomes and flow cytometry. Therefore, it proved that these methods are suitable, quick and easy methods for the identification of the ploidy level of F. multiflora.

Suspension Culture-Mediated Tetraploid Formation in Mouse Embryonic Stem Cells

  • Lee, Jae-Hee;Gong, Seung-Pyo;Lim, Jeong-Mook;Lee, Seung-Tae
    • Reproductive and Developmental Biology
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    • v.36 no.1
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    • pp.21-26
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    • 2012
  • Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained $in$ $vitro$ for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

Histopathological and DNA Content Analysis of a Dermal Sarcoma in the Soft-shelled Turtle Pelodiscus sinensis

  • Syasina Iraida Germogenovna;Hur Jun-Wook;Kim Eun-Mi;Park In-Seok
    • Fisheries and Aquatic Sciences
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    • v.9 no.3
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    • pp.107-114
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    • 2006
  • A dermal sarcoma was found in a freshwater, soft-shelled turtle Pelodiscus sinensis. The neoplasm consisted of proliferating fibrous tissue and extended from the dermis. The overlying epidermis was hyperplastic and partially folded. The deeper dermis and hypodermis contained three large, discrete necrotic foci of -10 mm diameter. Numerous eosinophilic granule cells and macro phages surrounded the necrotic areas. A mixed population of cells with nuclear pleomorphism was observed between the papillary layers of vessels. This area also had regions of different histological structures: (l) regularly arranged, spindle-shaped cells with compact nuclei in a fine-fibrillar matrix; (2) haphazardly arranged cells ($\leq$ 23 11m diameter) with ovoid, highly hypertrophic, faintly stained nuclei; and (3) cells (3.6-5.8 11m diameter) with irregularly shaped nuclei and marginal condensed chromatin in a myxomatous matrix. Some mitotic figures, binucleate cells, and multinucleate giant cells of up to 50 11m in length were also found. Flow cytometry of propidium iodide-stained cells yielded different histograms for the normal skin and the skin (primarily epidermis) and fibrous dermis of the tumor, indicating DNA heterogeneity in the dermal portion of the tumor. The ploidy indices for the dermal cells were 1.91 and 0.78, as compared to normal cells.

An efficient strategy for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis)

  • Nam, Yoon-Kwon;Park, Geyong-Cheol;Kim, Dong-Soo
    • Proceedings of the Korean Aquaculture Society Conference
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    • 2003.10a
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    • pp.38-38
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    • 2003
  • Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5$^{\circ}C$ for 1, 2 or 3 min followed by cold shock at $1.5^{\circ}C$ for 30, 45 or 60 min. When applied during the first cleavage metaphase of mud loach (Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7% and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

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Survivin protects fused cancer cells from cell death

  • Do, Mihyang;Kwak, In-Hae;Ahn, Ju-Hyun;Lee, In Jeong;Lee, Jae-Ho
    • BMB Reports
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    • v.50 no.7
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    • pp.361-366
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    • 2017
  • Tetraploidy, a potential precursor of cancer-associated aneuploidy, is produced either by cell fusion or failure of cytokinesis. In this study, low p53-expressing HeLa cells were used to address the fate of cancer cells after fusion. We found that massive cell death or growth arrest occurred a few days after fusion. Interestingly, cells with larger nuclei preferentially died after fusion, suggesting that a larger deviation of DNA content is a strong inducer of apoptosis. Notably, a fraction of cells escaped cell death. Also, the stability of survivin increased, and its localization changed preferentially to the cytosol in fused cells. Knockdown of survivin decreased the survival of fused cells, more than observed in unfused cells, showing increased dependency of fused cells on survivin. Collectively, after cancer cell fusion, some fused cells avoid the apoptotic crisis partly owing to survivin, and continue to proliferate, a process that contributes to human cancer progression.

Study on clinical chemistry and DNA ploidy pattern changes in carcinogenesis of the rat liver and kidney (간과 선장의 암유발과정에서 혈액화학효소 및 DNA ploidy pattern 의 변화에 대한 조사)

  • Jeong, Ja-Young;Jang, Dong-Deuk;Cho, Jae-Cheon;Lee, Yong-Soon
    • Korean Journal of Veterinary Pathology
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    • v.2 no.2
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    • pp.73-84
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    • 1998
  • This study was carried out to investigate on the serum chemistry and the DNA ploidy changes in carcinogenesis of the rat liver and kidney. Sixty male Sprague-Dawley rats were divided into two groups. Group I was non-treated control. Group II was given initiators (2,2'-dihydroxy- di-N-propylnitrosamine, 0.1% in drinking water(d.w.) for 1 week and N-ethyl-N-hydroxy-ethylnitrosamine; 0.15% in d.w. for 1 week) and promoters (3'methyl-cholanthrene; 3'MC, l0mg/kg, intraperitoneally(i.p.) twice a week and DL-serine; 0.05% in d.w. for 5 weeks, from 3 to 8 weeks). All examinations were performed at 12 and 20 weeks RBC, HGBCp<0.05) and PCVCp<0.01) significantly decreased in Group II at 20 weeks. Activities of ALT, AST(p<0.05) and GGT(p<0.01) were significantly increased in Group II at 20 weeks. Flow cytometric analysis showed hepatocyte nuclei from normal livers were predominantly tetraploid(66~67%) and then diploid(28~30%). Most of hepatocyte nuclei from carcinogen-treated rats were diploid (52~68%) and less were tetraploid(28~42%). Neoplastic liver nodules and hepatocellular carcinoma contained almost exclusively diploid nuclei. Renal cell nuclei from normal kidney were predominantly diploid(88~93%), those from carcinogen-treated rats had an abnormal DNA-content peak(aneuploidy, 6-7%), near the tetraploidy area. These results suggest that diploidy may be an effective screening marker of the liver carcinogenesis. Aneuploidy may be an useful marker in assessment of the experimental renal carcinogenesis.

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The effect of hydropriming and explants on shoot initiation and tetraploids in small watermelons

  • Phat, Phanna;Noh, Jae Jong;Ju, Ho-Jong;Lee, Gi-An;Ma, Kyung-Ho
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2017.06a
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    • pp.176-176
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    • 2017
  • Hydropriming had positive effects on the time for germination to reach 50%, the germination index, the time to final germination percentage, and the number of uniform seedlings with enlarged cotyledons in in vitro germination of small watermelon. In addition, the highest shoot initiation was obtained from hydroprimed cotyledonary nodes ($95{\pm}6%$), followed by non-primed cotyledonary nodes ($78{\pm}6%$), hydroprimed cotyledons ($72{\pm}4%$), and non-primed cotyledons ($48{\pm}4%$). Meanwhile, no shoots were initiated from hypocotyls. The total number of shoots that initiated from cotyledonary nodes and cotyledon explants was insignificant, indicating that both cotyledons and cotyledonary node were good sources for the in vitro culture. Choosing explant sources that favor tetraploidy should be the key for producing higher polyploidy plants; a total of 10.5% of tetraploid regenerants were entirely identified from cotyledon explants. Cotyledons with highly differentiated cells might show higher variations than cotyledonary nodes with more preexisting meristematic cells. Cells of cotyledon tissue might undergo changes in ploidy level during differentiation of the culture, or it might be that some of the variations were already present in the tissues of the donor plant. Morphological changes in fruit length of tetraploid regenerants are genotype-dependent.

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In vitro Induction of Tetraploid Roots by Various Pretreatments from Anther of Panax ginseng C. A. Meyer

  • Lee, Jung-Hye;Kim, Yu-Jin;Jung, Dae-Young;Shim, Ju-Sun;Kim, Ik-Hwan;Yang, Deok-Chun
    • Journal of Ginseng Research
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    • v.33 no.1
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    • pp.65-71
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    • 2009
  • This experiment was done to determine the optimum conditions for the induction of tetraploidy in Panax ginseng C. A. Meyer using bud length, temperature and plant growth regulator pretreatments. Highest callus formation was obtained when the medium was inoculated with flower bud in the size of 2-3 mm in length. The optimum temperature for the callus formation was high when treated at $4^{\circ}C$ for 4-5 days. Among the treatments of growth regulators and different concentration, highest callus formation was observed in combination of 5 mg/L 2,4-D and 1 mg/L kinetin for P. ginseng. As a result of flow cytometer analysis, all 7 adventitious roots were confirmed as tetraploidys. Cytological analysis revealed that the chromosome number of tetraploid roots was 96, while that of diploid roots was 48. Tetraploid ginseng roots were inoculated to flower bud size of 2-3 mm in length. The callus formation was optimum when treated with 1 mg/L 2,4-D at $4^{\circ}C$ for 5 days. Compared with control roots, tetraploid roots were thicker and longer and had few lateral branches. Fresh weight of tetraploid roots was relatively higher than the control roots.