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A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development
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作者 Lin Zhang Shi-Meng Cao +3 位作者 Hao Wu Meng Yan Jinsong Li Ling-Ling Chen 《Science China(Life Sciences)》 SCIE CAS CSCD 2024年第11期2297-2306,共10页
The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in liv... The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations. 展开更多
关键词 sperm-like stem cell semi-cloned technology CRISPR-RfxCas13d RNA knockdown embryonic development
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Fish germ cells 被引量:13
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作者 XU HongYan1, LI MingYou1, GUI JianFang2 & HONG YunHan1,2 1Department of Biological Sciences, National University of Singapore, Singapore 119260, Singapore 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China 《Science China(Life Sciences)》 SCIE CAS 2010年第4期435-446,共12页
Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and is... Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and isolated for culture and transplan-tation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking sperm to produce fertile offspring, upon nuclear being directly transferred into normal eggs. Such fish originated from a mosaic oocyte that had a haploid meiotic nucleus and a transplanted haploid mitotic cell culture nucleus. The first semi-cloned fish is Holly. Here we review the current status and future directions of understanding and manipulating fish germ cells in basic research and reproductive technology. 展开更多
关键词 FISH GERM cell GERM plasm REPRODUCTION REPRODUCTIVE technology semi-cloning
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Medaka fish stem cells and their applications 被引量:10
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作者 YI MeiSheng HONG Ni +4 位作者 LI ZhenDong YAN Yan WANG DanKe ZHAO HaoBin HONG YunHan 《Science China(Life Sciences)》 SCIE CAS 2010年第4期426-434,共9页
Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental pr... Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review. 展开更多
关键词 MEDAKA stem cells PLURIPOTENCY CHIMERA nuclear transplant semi-cloning
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Temporal regulation of prenatal embryonic development by paternal imprinted loci 被引量:6
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作者 Qing Li Yuanyuan Li +6 位作者 Qi Yin Shuo Huang Kai Wang Liangchai Zhuo Wei Li Boran Chang Jinsong Li 《Science China(Life Sciences)》 SCIE CAS CSCD 2020年第1期1-17,共17页
Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially ... Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development. 展开更多
关键词 imprinted loci semi-cloned technology temporal regulation H19-Igf2 Dlk1-Dio3 embryonic development
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Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells 被引量:2
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作者 Hongling Zhang Yuanyuan Li +4 位作者 Yongjian Ma Chongping Lai Qian Yu Guangyong Shi Jinsong Li 《Protein & Cell》 SCIE CSCD 2022年第2期102-119,共18页
The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,... The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints. 展开更多
关键词 paternal imprints androgenetic haploid ESCs DMRs semi-cloned mice alternative 2i
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