猪耳成纤维细胞转录组异质性及对核移植胚胎发育的潜在影响
收稿日期: 2020-06-22
修回日期: 2020-08-11
网络出版日期: 2020-08-31
基金资助
广东省乡村振兴战略专项(2018年)资助
Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation
Received date: 2020-06-22
Revised date: 2020-08-11
Online published: 2020-08-31
Supported by
Supported by Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry (2018)
同一来源的供体细胞之间存在异质性。许多研究已经表明体细胞核移植(somatic cell nuclear transfer, SCNT)效率与供体细胞有关。然而,鲜有在单细胞水平分析供体细胞异质性对核移植效率的潜在影响。本研究利用单细胞转录组测序技术对同一来源且随机挑选的52个猪耳组织成纤维细胞进行测序分析。结果表明有48个单细胞的基因表达模式相似,4个单细胞(编号为D11_1、D12_1、DW61_2和DW99_2)的基因表达模式与其他单细胞存在较大的差异,并且不存在基因表达模式完全相同的两个单细胞。以基因表达模式相似的48个单细胞作为对照,进一步分析了单细胞D11_1、D12_1、DW61_2和DW99_2的差异基因表达模式：首先利用R语言筛选4个单细胞的差异表达基因,并对前50差异表达基因进行汇总;然后对差异表达基因进行GO富集分析和KEGG通路分析。富集分析发现差异表达基因的主要分子功能包括能量代谢、蛋白质代谢和细胞对刺激的反应等;主要通路包括KEGG中富集的与细胞周期、细胞代谢、DNA复制相关的通路。根据以上研究结果并结合SCNT研究进展讨论了4个单细胞的差异基因表达模式对核移植胚胎发育效率的潜在影响。本研究揭示了猪耳组织成纤维细胞的转录组异质性,并提供了分析精英供体细胞的一种有效方法,为提高克隆效率带来新的思路。
周俊, 赵成成, 吴霄, 石俊松, 周荣, 吴珍芳, 李紫聪 . 猪耳成纤维细胞转录组异质性及对核移植胚胎发育的潜在影响[J]. 遗传, 2020 , 42(9) : 898 -915 . DOI: 10.16288/j.yczz.20-190
There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.
Key words： pigs; SCNT; donor cell; heterogeneity; single-cell sequencing
| [1] | Wilmut I, Schnieke AE, Mcwhir J, Kind AJ, Campbell KH . Viable offspring derived from fetal and adult mammalian cells. Nature, 1997,385(6619):810-813. |
| [2] | Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, de León FAP, Robl JM. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 1998,280(5367):1256-1258. |
| [3] | Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC . Pig cloning by microinjection of fetal fibroblast nuclei. Science, 2000,289(5482):1188-1190. |
| [4] | Liu Z, Cai YJ, Wang Y, Nie YH, Zhang CC, Xu YT, Zhang XT, Lu Y, Wang ZY, Poo M, Sun Q . Cloning of macaque monkeys by somatic cell nuclear transfer. Cell, 2018,172(4):881-887. |
| [5] | Beyhan Z, Iager AE, Cibelli JB . Interspecies nuclear transfer: implications for embryonic stem cell biology. Cell Stem Cell, 2007,1(5):502-512. |
| [6] | Niemann H, Lucas-Hahn A . Somatic cell nuclear transfer cloning: practical applications and current legislation. Reprod Domest Anim, 2012,47(Suppl. 5):2-10. |
| [7] | Tachibana M, Amato P, Sparman M, Gutierrez N M, Tippner-Hedges R, Ma H, Kang E, Fulati A, Lee H S, Sritanaudomchai H, Masterson K, Larson J, Eaton D, Sadler-Fredd K, Battaglia D, Lee D, Wu D, Jensen J, Patton P, Gokhale S, Stouffer RL, Wolf D, Mitalipov S . Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 2013,153(6):1228-1238. |
| [8] | Ao Z, Wu X, Zhou J, Gu T, Wang XW, Shi JS, Zhao CF, Cai GY, Zheng EQ, Liu DW, Wu ZF, Li ZC . Cloned pig fetuses exhibit fatty acid deficiency from impaired placental transport. Mol Reprod Dev, 2019,86(11):1569-1581. |
| [9] | Liu Y, Li J, L?vendahl P, Schmidt M, Larsen K, Callesen H . In vitro manipulation techniques of porcine embryos: a meta-analysis related to transfers, pregnancies and piglets. Reprod Fertil Dev, 2015,27(3):429-439. |
| [10] | Jang G, Park ES, Cho JK, Bhuiyan MM, Lee BC, Kang SK, Hwang WS . Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells. Theriogenology, 2004,62(3-4):512-521. |
| [11] | Zhang DF, Liu D, Tang LL, Wang Y, Chen Y, Wang K, Wang GL, Schellander K, Cailu L . Effects of different donor cells on the development of nuclear-trans-ferred porcine embryos. Hereditas(Beijing), 2007,29(2):211-217. |
| [11] | 张德福, 刘东, 汤琳琳, 王英, 陈茵, 王凯, 王根林 , KARL Schellander, LIN Cailu. 不同供体细胞及其处理对猪核移植重构胚体外发育的影响. 遗传, 2007,29(2):211-217. |
| [12] | Ruan ZY, Zhao X, Li ZD, Qin XL, Shao QM, Ruan QY, Deng YF, Jiang JR, Huang B, Lu FH, Shi DS . Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos. Reprod Domest Anim, 2019,54(1):11-22. |
| [13] | Rideout WR, Eggan K, Jaenisch R . Nuclear cloning and epigenetic reprogramming of the genome. Science, 2001,293(5532):1093-1098. |
| [14] | Yang XQ, Wu ZF, Li ZC . Advances in epigenetic reprogramming of somatic cells nuclear transfer in mammals. Hereditas(Beijing), 2019,41(12):1099-1109. |
| [14] | 杨旭琼, 吴珍芳, 李紫聪 . 哺乳动物体细胞核移植表观遗传重编程研究进展. 遗传, 2019,41(12):1099-1109. |
| [15] | Zhai YH, Li W, Zhang ZR, Cao YQ, Wang ZZ, Zhang S, Li ZY . Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs. Mol Reprod Dev, 2018,85(1):26-37. |
| [16] | Yamanaka S . Elite and stochastic models for induced pluripotent stem cell generation. Nature, 2009,460(7251):49-52. |
| [17] | Inoue K, Ogonuki N, Mochida K, Yamamoto Y, Takano K, Kohda T, Ishino F, Ogura A . Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol Reprod, 2003,69(4):1394-1400. |
| [18] | Zhou C, Zhang JC, Zhang M, Wang DB, Ma Y, Wang Y, Wang YZ, Huang YM, Zhang Y . Transcriptional memory inherited from donor cells is a developmental defect of bovine cloned embryos. Faseb J, 2020,34(1):1637-1651. |
| [19] | Picelli S, Faridani OR, Bj?rklund AK, Winberg G, Sagasser S, Sandberg R . Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc, 2014,9(1):171-181. |
| [20] | Grubman A, Chew G, Ouyang JF, Sun GZ, Choo XY, Mclean C, Simmons RK, Buckberry S, Vargas-Landin DB, Poppe D, Pflueger J, Lister R, Rackham O, Petretto E, Polo JM . A single-cell atlas of entorhinal cortex from individuals with Alzheimer's disease reveals cell-type- specific gene expression regulation. Nat Neurosci, 2019,22(12):2087-2097 |
| [21] | Tang FC, Barbacioru C, Wang YZ, Nordman E, Lee C, Xu NN, Wang XH, Bodeau J, Tuch BB, Siddiqui A, Lao K , Surani MA. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods, 2009,6(5):377-382. |
| [22] | Kim D, Langmead B, Salzberg SL . HISAT: a fast spliced aligner with low memory requirements. Nat Methods, 2015,12(4):357-360. |
| [23] | Wang LK, Feng ZX, Wang X, Wang XW, Zhang XG . DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 2010,26(1):136-138. |
| [24] | Young MD, Wakefield MJ, Smyth GK, Oshlack A . Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol, 2010,11(2):R14. |
| [25] | Robinson MD, Mccarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 2010,26(1):139-140. |
| [26] | Navin N, Hicks J . Future medical applications of single-cell sequencing in cancer. Genome Med, 2011,3(5):31. |
| [27] | Novick A, Weiner M . Enzyme induction as an all-or-none phenomenon. Proc Natl Acad Sci USA, 1957,43(7):553-566. |
| [28] | Coskun AF, Eser U, Islam S . Cellular identity at the single-cell level. Mol Biosyst, 2016,12(10):2965-2979. |
| [29] | Menon M, Mohammadi S, Davila-Velderrain J, Goods BA, Cadwell TD, Xing Y, Stemmer-Rachamimov A, Shalek AK, Love JC, Kellis M, Hafler BP . Single-cell transcriptomic atlas of the human retina identifies cell types associated with age-related macular degeneration. Nat Commun, 2019,10(1):4902. |
| [30] | Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007,131(5):861-872. |
| [31] | Lowry WE, Richter L, Yachechko R, Pyle AD, Tchieu J, Sridharan R, Clark AT, Plath K . Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA, 2008,105(8):2883-2888. |
| [32] | Polo JM, Liu S, Figueroa M E, Kulalert W, Eminli S, Tan KY, Apostolou E, Stadtfeld M, Li YS, Shioda T, Natesan S, Wagers AJ, Melnick A, Evans T, Hochedlinger K . Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol, 2010,28(8):848-855. |
| [33] | Lai L, Tao T, Macháty Z, Kühholzer B, Sun QY, Park KW, Day BN, Prather RS . Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors. Biol Reprod, 2001,65(5):1558-1564. |
| [34] | Chesné P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP . Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol, 2002,20(4):366-369. |
| [35] | Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G . Ubiquitin tag for sperm mitochondria. Nature, 1999,402(6760):371-372. |
| [36] | Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC . Pig cloning by microinjection of fetal fibroblast nuclei. Science, 2000,289(5482):1188-1190. |
| [37] | Gupta S, Sahu D, Bomalaski JS, Frank I, Boorjian SA, Thapa P, Cheville J C, Hansel DE . Argininosuccinate Synthetase-1 (ASS1) loss in high-grade neuroendocrine carcinomas of the urinary bladder: implications for targeted therapy with ADI-PEG 20. Endocr Pathol, 2018,29(3):236-241. |
| [38] | Moren L, Perryman R, Crook T, Langer JK, Oneill K, Syed N, Antti H . Metabolomic profiling identifies distinct phenotypes for ASS1 positive and negative GBM. BMC Cancer, 2018,18(1):167. |
| [39] | Bang J, Lee E, Lee AR, Il Lee J, Choi SH, Seol D, Park C, Lee DR . The effect of cell penetrating peptide-conjugated coactivator-associated arginine methyltransferase 1 (CPP-CARM1) on the cloned mouse embryonic development. Sci Rep, 2018,8(1):16721. |
| [40] | Abdel-Hady AE, Beige J, Kreutz R, Bolbrinker J . Effect of UMOD genotype on long-term graft survival after kidney transplantation in patients treated with cyclosporine-based therapy. Pharmacogenomics J, 2018,18(2):227-231. |
| [41] | Bailie C, Kilner J, Maxwell AP, Mcknight AJ . Development of next generation sequencing panel for UMOD and association with kidney disease. PLoS One, 2017,12(6):e0178321. |
| [42] | Davis D, Kannan M, Wattenberg B . Orm/ORMDL proteins: Gate guardians and master regulators. Adv Biol Regul, 2018,70:3-18. |
| [43] | Fava RA, Browning JL, Gatumu M, Skarstein K, Bolstad AI . LTBR-pathway in Sjogren's syndrome: CXCL13 levels and B-cell-enriched ectopic lymphoid aggregates in NOD mouse lacrimal glands are dependent on LTBR. Adv Exp Med Biol, 2011,691:383-390. |
| [44] | Zhu QQ, Li N, Li F, Sang J, Deng H, Han QY, Lv Y, Li CY, Liu ZW . Association of LTBR polymorphisms with chronic hepatitis B virus infection and hepatitis B virus-related hepatocellular carcinoma. Int Immunopharmacol, 2017,49:126-131. |
| [45] | Sia D, Losic B, Moeini A, Cabellos L, Hao K, Revill K, Bonal D, Miltiadous O, Zhang ZY, Hoshida Y, Cornella H, Castillo-Martin M, Pinyol R, Kasai Y, Roayaie S, Thung S N, Fuster J, Schwartz ME, Waxman S, Cordon-Cardo C, Schadt E, Mazzaferro V, Llovet JM . Massive parallel sequencing uncovers actionable FGFR2-PPHLN1 fusion and ARAF mutations in intrahepatic cholangiocarcinoma. Nat Commun, 2015,6:6087. |
| [46] | Chung YG, Matoba S, Liu YT, Eum JH, Lu FL, Jiang W, Lee JE, Sepilian V, Cha KY, Lee DR, Zhang Y . Histone demethylase expression enhances human somatic cell nuclear transfer efficiency and promotes derivation of pluripotent stem cells. Cell Stem Cell, 2015,17(6):758-766. |
| [47] | Boone PM, Yuan B, Gu S, Ma ZW, Gambin T, Gonzaga-Jauregui C, Jain M, Murdock TJ, White JJ, Jhangiani SN, Walker K, Wang QY, Muzny DM, Gibbs RA, Hejtmancik JF, Lupski JR, Posey JE, Lewis RA . Hutterite-type cataract maps to chromosome 6p21.32- p21.31, cosegregates with a homozygous mutation in LEMD2, and is associated with sudden cardiac death. Mol Genet Genomic Med, 2016,4(1):77-94. |
| [48] | Mcgee LJ, Jiang AL, Lan Y. Golga5 is dispensable for mouse embryonic development and postnatal survival.Genesis, 2017, 55(7): 10. 1002/dvg. 23039. |
| [49] | O'Grady GL, Best HA, Sztal TE, Schartner V, Sanjuan-Vazquez M, Donkervoort S, Abath NO, Sutton RB, Ilkovski B, Romero NB, Stojkovic T, Dastgir J, Waddell L B, Boland A, Hu Y, Williams C, Ruparelia AA, Maisonobe T, Peduto AJ, Reddel SW, Lek M, Tukiainen T, Cummings BB, Joshi H, Nectoux J, Brammah S, Deleuze JF, Ing VO, Ramm G, Ardicli D, Nowak KJ, Talim B, Topaloglu H, Laing NG, North K N, Macarthur DG, Friant S, Clarke NF, Bryson-Richardson RJ, B?nnemann CG, Laporte J, Cooper ST,. Variants in the oxidoreductase PYROXD1 cause early-onset myopathy with internalized nuclei and myofibrillar disorganization. Am J Hum Genet, 2016,99(5):1086-1105. |
| [50] | Al-Dabbagh N, Al-Shahrani H, Al-Dohayan N, Mustafa M, Arfin M, Al-Asmari AK . The SPARC-related modular calcium binding protein 2 (SMOC2) gene polymorphism in primary glaucoma: a case-control study. Clin Ophthalmol, 2017,11:549-555. |
| [51] | Huang XQ, Zhou ZQ, Zhang XF, Chen CL, Tang Y, Zhu Q, Zhang JH, Xia JC . Overexpression of SMOC2 attenuates the tumorigenicity of hepatocellular carcinoma cells and is associated with a positive postoperative prognosis in human hepatocellular carcinoma. J Cancer, 2017,8(18):3812-3827. |
| [52] | Hagan N, Guarente J, Ellisor D, Zervas M . The temporal contribution of the Gbx2 lineage to cerebellar neurons. Front Neuroanat, 2017,11:50. |
| [53] | Mallika C, Guo QX, Li JYH . Gbx2 is essential for maintaining thalamic neuron identity and repressing habenular characters in the developing thalamus. Dev Biol, 2015,407(1):26-39. |
| [54] | Alber M, Kalscheuer VM, Marco E, Sherr E, Lesca G, Till M, Gradek G, Wiesener A, Korenke C, Mercier S, Becker F, Yamamoto T, Scherer SW, Marshall CR, Walker S, Dutta UR, Dalal A B, Suckow V, Jamali P, Kahrizi K, Najmabadi H, Minassian BA . ARHGEF9 disease: Phenotype clarification and genotype-phenotype correlation. Neurol Genet, 2017,3(3):e148. |
| [55] | Klein KM, Pendziwiat M, Eilam A, Gilad R, Blatt I, Rosenow F, Kanaan M, Helbig I, Afawi Z . The phenotypic spectrum of ARHGEF9 includes intellectual disability, focal epilepsy and febrile seizures. J Neurol, 2017,264(7):1421-1425. |
| [56] | Li HS, Song MM, Yang W, Cao P, Zheng L, Zuo YC . A Comparative analysis of single-cell transcriptome identifies reprogramming driver factors for efficiency improvement. Molecular Therapy - Nucleic Acids, 2020,19:1053-1064. |
| [57] | Inoue K, Ogonuki N, Mochida K, Yamamoto Y, Takano K, Kohda T, Ishino F, Ogura A . Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol Reprod, 2003,69(4):1394-1400. |
| [58] | Xie BT, Zhang H, Wei RY, Li QN, Weng XG, Kong QR, Liu ZH . Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming. Reproduction, 2016,151(1):9-16. |
| [59] | Matoba S, Liu YT, Lu FL, Iwabuchi KA, Shen L, Inoue A, Zhang Y . Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell, 2014,159(4):884-895. |
| [60] | Liu Y, Wu FR, Zhang L, Wu XQ, Li DK, Xin J, Xie J, Kong F, Wang WY, Wu QQ, Zhang D, Wang R, Gao SR, Li WY . Transcriptional defects and reprogramming barriers in somatic cell nuclear reprogramming as revealed by single-embryo RNA sequencing. BMC Genomics, 2018,19(1):734. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
