Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (2): 400-411.doi: 10.3864/j.issn.0578-1752.2021.02.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

CRISPR/Cas9 Mediated Exogenous Gene Knock-in at ROSA26 Locus in Sheep Umbilical Cord Mesenchymal Stem Cells

LI SongMei(),QIU YuGe,CHEN ShengNan,WANG XiaoMeng,WANG ChunSheng()   

  1. College of Life Science,Northeast Forestry University, Harbin 150040
  • Received:2020-02-23 Accepted:2020-05-13 Online:2021-01-16 Published:2021-02-03
  • Contact: ChunSheng WANG E-mail:Li@outlook.com;wangchunsheng79@163.com

RichHTML

11

PDF

252

Abstract:

【Objective】The use of CRISPR/Cas9 system to establish a sheep tracing umbilical cord mesenchymal stem cell line lays the foundation for the clinical therapy and mechanism of MSCs. 【Method】Three single guide RNAs (sgRNA)were designed and synthesized for the ROSA26 locus of sheep by using the online tool ZiFiT Targeter Version 4.2, PCR was performed using the point mutation method with the px330 plasmid as a template. The PCR product was circularized after the plasmid DNA was removed by Dpn I, the sgRNA / Cas9 vector targeting sROSA26 was constructed after enzyme digestion and sequencing identification. The constructed plasmid contained Cas9 and sgRNA expression cassettes, which were driven by the U6 promoter. Because of the off-target effect of CRISPR/Cas9 system, the vector was transfected into sUMSCs, after extraction of its genome for PCR, T7E1 enzyme digestion. Then analyzing the gray levels of the bands to detect the vector editing activity. The sgRNA / Cas9 vector can cuts the double-stranded DNA at the target site. In order to knock in the reporter gene, the Donor vector needs to repair the sequence on the donor DNA by homologous recombination at the DNA break to insert target sequence. Based on the sgRNA sequence, the left and right homology arm amplification primers were designed and synthesized at the upstream and downstream of the sROSA26 locus. Using sheep's whole genome as a template for PCR amplification to obtain left and right homology arms. After recovery and purification, recombinant with pMD19-Simple to obtain left and right homologous recombinant plasmids. The left and right homology arm plasmids were ligated with the Donor expression vector DC-DON-SH02 ROSA26 to obtain the left arm recombinant targeting vector. The homologous recombination vector carrying green fluorescent protein (GFP) and puromycin resistance gene was constructed. The survival time of cells with different concentrations of puromycin was observed in well-growing sUMSCs to determine the optimal concentration and time of resistance screening. In sUMSCs, sgRNA / Cas9 vector and Donor vector were co-transfected by liposome method. Puromycin resistance screening was performed 48 hours after transfection. After the screening, cells need to be replaced with normal media and continue to expand. Observing the expression of GFP and extracting the positive cell genome , designing two pairs of upstream and downstream primers for ROSA26 site for PCR to detect its editing status. 【Result】(1) Three sgRNA primers were designed for the sheep ROSA26 locus. The expression vector px330-sgRNA1/2/3 were successfully constructed by point mutation method and the vectors were transfected into sheep umbilical cord mesenchymal stem cells. T7E1 enzyme assay results indicated that the editing efficiency of px330-sgRNA1 was the highest, about 20%. (2) Based on sgRNA1, the left and right homology arms of the targeting vector were obtained by PCR, and the recombinant vector(sROSA26-HA) of sheep ROSA26 locus was successfully obtained by a series of molecular biological methods; (3) px330-sgRNA1 and sROSA26-HA were co-transfected into sheep umbilical cord mesenchymal stem cells using Lipofectamine2000, and positive colonies could express green fluorescent protein were obtained after 1.5 μg·mL -1 puromycin selection for 15 days. PCR detection of one clone indicated that the targeting vector had been integrated into the genome. 【Conclusion】The exogenous GFP gene could successfully integrated in specific locus of sheep umbilical cord mesenchymal stem cells using the CRISPR/Cas9 system, and these cells can be used to trace the position and differentiated cell type of MSCs. This research laid a foundation for further clinical transformation of mesenchymal stem cells.

Key words: sheep, CRISPR/Cas9, homologous recombination, sheep umbilical cord mesenchymal stem cells, GFP, ROSA26

Table 1

Sequences and product length of sgRNA and primers"

sgRNA/引物 sgRNA/Primers 序列 Oligonucleotide sequences 片段 Length (bp)
KOD-R 5′-GGTGTTTCGTCCTTTCCAC 19
px330-sgRNA-1 5′-CCAGCAGGTATAAGATTTAGGTTTTAGAGCTAGAATAGCAGGT 43
px330-sgRNA-2 5′-CCTCTAAATCTTATACCTGCGTTTTAGAGCTAGAATAGCAGGT 43
px330-sgRNA-3 5′-TGTCCTGCAGTGGATCCAGCGTTTTAGAGCTAGAATAGCAGGT 43
px330-TP-F 5′-GCTGCCTGAAGGACAAGACTA 21
px330-TP-R 5′-GGCAACACCTGGGACTGATTT 21
sROSA26-LHA-F 5′-ACTAGTTACGCTGAAAGGGAAAGAGG 26
sROSA26-LHA-R 5′-ACGCGTAGGACAACGCCCAGGATT 24
sROSA26-RHA-F 5′-TGTACACCCAATTTCTTTATCTTCCC 26
sROSA26-RHA-R 5′-ACATGTTCCTGTCAGTAGTTACCACCC 27
SRL-F 5′-AAGAGGCTGTGCTCTGGG 18
SRL-R 5′-CGTGAGTCAAACCGCTATCC 20
SRR-F 5′-TCCCTAAAGAAACAGTGGC 19
SRR-R 5′-CACGTTTGTGATGATGGAAT 20

Fig. 1

The BbsⅠenzyme identification of sROSA26-sgRNA-1 1: Trans8k DNA Marker; 2: Plasmid PX330; 3: Enzyme identification of restructuring plasmid"

Fig. 2

Transfection efficiency A：sUMSCs;B：sUMSCs After transfecion;C：Control group sUMSCs（50×） (A: the sUMSCs cell line after transfecting empty plasmid as a positive control; B, C:Transfer sROSA26-sgRN-1/2/3 vectors with GFP into sUMSCs cell lines using Trim Away technique, capture the bright and dark field after transfection for 48h, and the transfection efficiency was determined according to the observed fluorescence intensity.)"

Fig. 3

T7E1 digestion identification 1: Trans2K DNA Marker; 2: Wild type PCR product; 3: Wild type T7E1 digestion product; 4: Mutated T7E1 digestion product"

Fig. 4

Left homology arm PCR amplification 1: Left arm PCR product; 2: Trans8K DNA Marker"

Fig. 5

Right homology arm PCR amplification 1: Right arm PCR product; 2: Trans8K DNA Marker"

Fig. 6

Left arm cloning vector digestion identification 1: D2000Plus DNA Marker; 2: Identification of the left arm cloning"

Fig7.

Right arm cloning vector digestion identification 1: Identification of right arm cloning; 2: Trans8K DNA Marker"

Fig. 8

Left arm expression vector enzyme digestion identification 1: Trans8K DNA Marker; 2: Left arm expression vector enzyme digestion identification"

Fig. 9

sROSA26-HA enzyme digestion identification 1: D2000Plus DNA Marker; 2: sROSA26-HA enzyme digestion identification"

Fig. 10

Donor vector construction flowchart"

Fig. 11

Construction of the DC-DON-SH02 ROSA26 vector"

Fig. 12

Select the best puromycin concentration A: sUMSCs（50×）; B: After 1.5μg·mL-1 puromycin screening for 48h sUMSCs（50×）; C: At the end of puromycin screening sUMSCs（50×） (Add puromycin to sUMSCs when the confluence was 70%-80%, and record the time and concentration of almost all cell death as the foundation for subsequent resistance screening. )"

Fig. 13

Screening of best puromycin concentration statistics"

Fig. 14

Cloning and screening of positive cells A: After puromycin screening for 16d sUMSC-1（Bright field 50×）; B: After puromycin screening for 16d sUMSC（Dark field 50×） (Transfect the sROSA26-HA vector with GFP into the sUMSCs cell line, add puromycin after transfecion 24h, most cells express green fluorescence after 16 days of screening.After that, change to normal medium.)"

Fig. 15

Positive cells were further cultured A: After puromycin screening for 16d sUMSCs（Bright field 50×）; B: After puromycin screening for 16d sUMSCs（Dark field 50×） (Subculture the screened positive clones to obtain sufficient cell numbers for subsequent experimental studies)"

Fig. 16

Upstream PCR identification of homologous recombination 1: D2000 DNA Marker; 2: Homologous recombination upstream PCR product; 3: Wild type PCR product"

Fig. 17

Downstream PCR identification of homologous recombination 1: D2000 DNA Marker; 2: Homologous recombination downstream PCR product; 3: Wild type PCR product"

[1] KIM H, KIM J S. A guide to genome engineering with programmable nucleases. Nature Reviews Genetics, 2014,15:321-334.
doi: 10.1038/nrg3686 pmid: 24690881
[2] MALI P, YANG L, ESVELT K M, AACH J, GUELL M, DICARLO J E, NORVILLE J E, CHURCH G M. RNA-guided human genome engineering via Cas9. Science, 2013,339(6121):823-826.
pmid: 23287722
[3] SHEN B, ZHANG J, WU H, WANG J, MA K, LI Z, ZHANG X, ZHANG P, HUANG X. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013,23:720-723.
doi: 10.1038/cr.2013.46 pmid: 23545779
[4] ZHAO P, ZHANG Z, KE H, YUE Y, XUE D. Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system. Cell Research, 2014,24:247-250.
doi: 10.1038/cr.2014.9 pmid: 24418757
[5] LI D, QIU Z, SHAO Y, CHEN Y, GUAN Y, LIU M, LI Y, GAO N, WANG L, LU X, ZHAO Y, LIU M. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nature Biotechnology, 2013,31:681-683.
doi: 10.1038/nbt.2661 pmid: 23929336
[6] HWANG W Y, FU Y, REYON D, MAEDER M L, TSAI S Q, SANDER J D, PETERSON R T, YEH J R, JOUNG J K. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology, 2013,31:227-229.
pmid: 23360964
[7] CHEN Y, ZHENG Y, KANG Y, YANG W, NIU Y, GUO X, TU Z, SI C, WANG H, XING R, PU X, YANG S H, LI S, JI W, LI X J. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Human Molecular Genetics, 2015,24:3764-3774.
pmid: 25859012
[8] SU X P, CUI K Q, DU S S, LI H L, LU F H, SHI D S, LIU Q Y. Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. In Vitro Cellular and Developmental Biology - Animal, 2018,54(5):375-383.
[9] WU M, WEI C, LIAN Z, LIU R, ZHU C, WANG H, CAO J, SHEN Y, ZHAO F, ZHANG L, MU Z, WANG Y, WANG X, DU L, WANG C. ROSA26-targeted sheep gene knock-in via CRISPR-Cas9 system. Scientific Reports, 2016,5:24360.
[10] 黄里, 陈小, 项鹏. 间充质干细胞临床转化:进展与挑战. 中国细胞生物学学报, 2018,40(13):2226-2236.
HUANG L, CHEN X, XIANG P. Clinical transformation of mesenchymal stem cells: Progress and challenges. Chinese Journal of Cell Biology, 2018,40(13):2226-2236. (in Chinese)
[11] RIPA R S, HAACK-SØRENSEN M, WANG Y, JØRGENSEN E, MORTENSEN S, BINDSLEV L, FRIIS T, KASTRUP J. Bone marrow derived mesenchymal cell mobilization by granulocyte- colony stimulating factor after acute myocardial infarction: results from the Stem Cells in Myocardial Infarction (STEMMI) trial. Circulation, 2007,116:124-130.
[12] CAPLAN A I. Adult mesenchymal stem cells: when, where, and how. Stem Cells International. 2015,DOI: 10.1155/2015/628767.
doi: 10.1155/2020/8857057 pmid: 33424980
[13] RINGE J, STRASSBURG S, NEUMANN K, ENDRES M, NOTTER M, BURMESTER GR, KAPS C, SITTINGER M. Towards in situ tissue repair: Human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2. and migrate upon stimulation with CXCL8 but not CCL2. Journal of Cellular Biochemistry, 2007,101(1):135-146.
doi: 10.1002/jcb.21172 pmid: 17295203
[14] SAVKOVIC V, LI H, SEON J K, HACKER M, FRANZ S, SIMON J C. Mesenchymal stem cells in cartilage regeneration. Current Stem Cell Research and Therapy, 2014,9(6):469-488.
[15] 杨汉华, 田树凤, 谢丽春, 陈运彬, 马廉. 经鼻腔移植人脐带间充质干细胞修复新生大鼠缺氧缺血性脑损伤. 中国组织工程研究, 2019,23(21):3386-3391.
YANG H H, TIAN S F, XIE L C, CHEN Y B, MA L. Repair of hypoxic ischemic brain injury in newborn rats by nasal transplantation of human umbilical cord mesenchymal stem cells. China Tissue Engineering Research, 2019,23(21):3386-3391. (in Chinese)
[16] KRAMPERA M. Mesenchymal stromal cell ‘licensing’: a multistep process. Leukemia, 2011,25(9) : 1408-1414.
pmid: 21617697
[17] ELJAAFARI A, TARTELIN M L, AISSAOUI H, CHEVREL G, OSTA B, LAVOCAT F, MIOSSEC P. Bone marrow-derived and synovium‐derived mesenchymal cells promote Th17 cell expansion and activation through caspase 1 activation: Contribution to the chronicity of rheumatoid arthritis. Arthritis and Rheumatism, 2012,64(7). DOI: 10.1002/art.34391.
doi: 10.1002/art.34401 pmid: 22275298
[18] LIANG J, ZHANG H, HUA B, WANG H, LU L, SHI S, HOU Y, ZENG X, GILKESON G S, SUN L. Allogenic mesenchymal stem cells transplantation in refractory systemic systemic lupus erythematosus: a pilot clinical study. Annals of the Rheumatic Diseases, 2010,69(8):1423-1429.
doi: 10.1136/ard.2009.123463 pmid: 20650877
[19] WANG D, ZHANG H, LIANG J, LI X, FENG X, WANG H, HUA B, LIU B, LU L, GILKESON G S, SILVER R M, CHEN W, SHI S, SUN L. Allogeneic mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus: 4 years of experience. Cell Transplantation, 2013,22(12):2267-2277.
pmid: 24388428
[20] WANG Y, CHEN X, CAO W, SHI Y. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nature Immunology. 2014,15(11):1009-1016.
pmid: 25329189
[21] BIANCO P, ROBEY P G, SIMMONS P J. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell, 2008,2(4):313-319.
doi: 10.1016/j.stem.2008.03.002 pmid: 18397751
[22] SPASOVSKI D, SPASOVSKI V, BASCAREVIC Z, STOJILJKOVIC M, VRECA M, ANĐELKOVIC M, PAVLOVIC S. Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis. The Journal of Gene Medicine, 2018,20(1) : 1357-1366.
[23] ZHANG J P, LI X L, LI G H, CHEN W, ARAKAKI C, BOTIMER G D, BAYLINK D, ZHANG L, WEN W, FU YW, XU J, CHUN N, YUAN W, CHENG T, ZHANG X B. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biology, 2017,18(1):35.
doi: 10.1186/s13059-017-1164-8 pmid: 28219395
[24] YAO X, WANG X, HU X, LIU Z, LIU J, ZHOU H, SHEN X, WEI Y, HUANG Z, YING W, WANG Y, NIE Y H, ZHANG C C, LI S, CHENG L, WANG Q, WU Y, HUANG P, SUN Q, SHI L, YANG H. 2017b. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Research, 2017,27(6):801-814.
pmid: 28524166
[25] BARRANGOU R, FREMAUX C, DEVEAU H, RICHARDS M, BOYAVAL P, MOINEAU S, ROMERO D A, HORVATH P. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007,315(5819):1709-1712.
doi: 10.1126/science.1138140 pmid: 17379808
[26] STERNBERG S H, BENJAMIN L F, MATIAS K, DOUDNA J A. Conformational control of DNA arget cleavage by CRISPR-Cas9. Nature, 2015,527(7576):110-113.
pmid: 26524520
[27] DEKELVER R C, CHOI V M, MOEHLE E A, PASCHON D E, HOCKEMEYER D, MEIJSING S H, SANCAK Y, CUI X, STEINE E J, MILLER J C, TAM P, BARTSEVICH V V, MENG X, RUPNIEWSKI I, GOPALAN S M, SUN H C, PITZ K J, ROCK J M, ZHANG L, DAVIS G D, REBAR E J, CHEESEMAN I M, YAMAMOTO K R, SABATINI D M, JAENISCH R, GREGORY P D, URNOV F D. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Research, 2010,20(8):1133-1142.
pmid: 20508142
[28] LI X, YANG Y, BU L, GUO X, TANG C, SONG J, FAN N, ZHAO B, OUYANG Z, LIU Z, ZHAO Y, YI X, QUAN L, LIU S, YANG Z, OUYANG H, CHEN YE, WANG Z, LAI L. ROSA26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Research, 2014,24(4):501-504.
doi: 10.1038/cr.2014.15 pmid: 24503648
[29] KONG Q, HAI T, MA J, HUANG T, JIANG D, XIE B, WU M, WANG J, SONG Y, WANG Y, HE Y, SUN J, HU K, GUO R, WANG L, ZHOU Q, MU Y, LIU Z. ROSA26 locus supports tissue-specific promoter driving transgene expression specifically in pig. PLoS One, 2014,9(9):e107945.
doi: 10.1371/journal.pone.0107945 pmid: 25232950
[30] FRIEDRICH G, SORIANO P. Promoter traps in embryonic stem cell: a genetic screen to identify and mutate developmental genes in mice. Genes and Development, 1991,5(9):1513-1523.
doi: 10.1101/gad.5.9.1513 pmid: 1653172
[31] PLATT R J, CHEN S, ZHOU Y, YIM M J, SWIECH L, KEMPTON H R, DAHLMAN J E, PARNAS O, EISENHAURE T M, JOVANOVIC M, GRAHAM D B, JHUNJHUNWALA S, HEIDENREICH M, XAVIER R J, LANGER R, ANDERSON D G, HACOHEN N, REGEV A, FENG G, SHARP P A, ZHANG F. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 2014,159(2):440-455.
doi: 10.1016/j.cell.2014.09.014 pmid: 25263330
[32] SHIMOMURA O. Structure of the chromophore of Aequorea green fluorescent protein. FEBS Letters, 1979,104(2):220-222.
[33] GARNEAU J E, DUPUIS M, VILLION M, ROMERO D A, BARRANGOU R, BOYAVAL P, FREMAUX C, HORVATH P, MAGADÁN A H, MOINEAU S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010,468(7320):67-71.
pmid: 21048762
[34] STERNBERG S H, SY R, MARTIN J, GREENE E C, DOUDNA J A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 2014,507(7490):62-67.
doi: 10.1038/nature13011 pmid: 24476820
[1] LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(9): 1868-1876.
[2] CHE DaLu,ZHAO LiChen,CHENG SuCai,LIU AiYu,LI XiaoYu,ZHAO ShouPei,WANG JianCheng,WANG Yuan,GAO YuHong,SUN XinSheng. Effect of Litter Bed on Growth Performance and Odor Emission in Fattening Lamb [J]. Scientia Agricultura Sinica, 2022, 55(24): 4943-4956.
[3] SONG ShuZhen, GAO LiangShuang, LI Hong, GONG XuYin, LIU LiShan, WEI YuBing. Effects of Feeding Levels on Muscle Tissue Structure and Muscle Fiber Composition Related Genes in Sheep [J]. Scientia Agricultura Sinica, 2022, 55(21): 4304-4314.
[4] ChunTao ZHANG,Tao MA,Yan TU,QiYu DIAO. Effects of Circadian Rhythm on Rumen Fermentation and Nutrient Digestion of Mutton Sheep [J]. Scientia Agricultura Sinica, 2022, 55(18): 3664-3674.
[5] LIU WangJing,TANG DeFu,AO ChangJin. Effect of Allium mongolicum Regel and Its Extracts on the Growth Performance, Carcass Characteristics, Meat Quality and Serum Biochemical Indices of Captive Small-Tailed Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(17): 3461-3472.
[6] LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen. Association Analysis of the ADIPOQ Variation with Sheep Growth Traits [J]. Scientia Agricultura Sinica, 2022, 55(11): 2239-2256.
[7] KE Na,HAO ZhiYun,WANG JianQing,ZHEN HuiMin,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin,ZHAO ZhiDong,HUANG ZhaoChun,LIANG WeiWei,WANG JiQing. The miR-221 Inhibits the Viability and Proliferation of Ovine Mammary Epithelial Cells by Targeting IRS1 [J]. Scientia Agricultura Sinica, 2022, 55(10): 2047-2056.
[8] YANG Min,XU HuaWei,WANG CuiLing,YANG Hu,WEI YueRong. Using CRISPR/Cas9-mediated Targeted Mutagenesis of ZmFKF1 Delayed Flowering Time in Maize [J]. Scientia Agricultura Sinica, 2021, 54(4): 696-707.
[9] WANG Qian,LI Zheng,ZHAO ShanShan,QIE MengJie,ZHANG JiuKai,WANG MingLin,GUO Jun,ZHAO Yan. Application of Stable Isotope Technology in the Origin Traceability of Sheep [J]. Scientia Agricultura Sinica, 2021, 54(2): 392-399.
[10] WU ShiYang,YANG XiaoYi,ZHANG YanWen,HOU DianYun,XU HuaWei. Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology [J]. Scientia Agricultura Sinica, 2021, 54(18): 3805-3817.
[11] WANG Chen,ZHANG HongWei,WANG HuCheng,SUN XiaoPing,LI FaDi,YANG BoHui. Energy and Protein Requirements of Alpine Merino Growing Sheep [J]. Scientia Agricultura Sinica, 2021, 54(16): 3537-3548.
[12] WANG JiQing,HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin. Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep [J]. Scientia Agricultura Sinica, 2021, 54(14): 3113-3123.
[13] ZHANG Wei,WANG ShiYin,GAO Li,YANG LiWei,DENG ShuangYi,LIU XiaoNa,SHI GuoQing,GAN ShangQuan. Investigation of miR-486 Target Genes in Skeletal Muscle of Bashbay Sheep in Different Development Periods [J]. Scientia Agricultura Sinica, 2021, 54(14): 3134-3148.
[14] LI ZhaoWei,LING DongLan,SUN CongYing,ZENG HuiLing,LIU KaiJi,LAN YingShan,FAN Kai,LIN WenXiong. CRISPR/Cas9 Targeted Editing of OsIAA11 in Rice [J]. Scientia Agricultura Sinica, 2021, 54(13): 2699-2709.
[15] LI RunTing,CHEN LongXin,ZHANG LiMeng,HE HaiYing,WANG Yong,YANG RuoChen,DUAN ChunHui,LIU YueQin,WANG YuQin,ZHANG YingJie. Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts [J]. Scientia Agricultura Sinica, 2021, 54(11): 2434-2444.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd