Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex

轮藻单细胞，新月藻属peracerosum-strigosum-littorale复合体的基因沉默转化子的制备

JA Jun Abe
NH Naoko Hirano
AK Ayumi Komiya
NK Naho Kanda
AF Anri Fujiwara
SH Sachie Hori
YT Yuki Tsuchikane
HS Hiroyuki Sekimoto

发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1813 浏览次数: 9416

评审: Maria SinetovaChristian SailerAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Plant & Cell Physiology, featuring study using the protocol.

Jul 2015

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

PEG介导的稳定的<em>Cyanidioschizon merolae</em>细胞核和叶绿体转化

PEG介导的稳定的Cyanidioschizon merolae细胞核和叶绿体转化

Maksymilian Zienkiewicz [...] Kinga Kania

2019年09月05日 4271 阅读

利用本生烟叶片基因瞬时表达或沉默研究青枯菌毒力的快速简便方法

利用本生烟叶片基因瞬时表达或沉默研究青枯菌毒力的快速简便方法

Wenjia Yu and Alberto P. Macho

2021年08月05日 3952 阅读

农杆菌介导的棉花遗传转化和通过体细胞胚胎发生的再生

农杆菌介导的棉花遗传转化和通过体细胞胚胎发生的再生

Alka Srivastava [...] Praveen C. Verma

2023年05月20日 3080 阅读

Abstract

To prepare the knockdown transformants of the Closterium peracerosum-strigosum-littorale (C. psl.) complex, particle bombardment was applied with a newly constructed vector (pSA0104) with an endogenous constitutive promoter fused to a DNA fragment corresponding to an antisense strand of a target gene. Using a hygromycin resistance gene (aph7”), hygromycin-resistant colonies were selected. After the second screening, integration of the vector into the genome was checked by PCR and the knockdown effect was evaluated by Western blotting using a specific antibody against the target protein.

Keywords: Knockdown (击倒) search Alga (藻类) search Closterium (新月藻) search Charophycean (charophycean) search Transformation (转型) search

Materials and Reagents

  1. 0.6 µm gold microcarriers (Bio-Rad Laboratories, catalog number: 165-2262 ) or 0.25 µm gold nanoparticle (BBI Solutions, catalog number: EMGC250 )
  2. Cell culture dishes, 90 mm x 20 mm polystyrene (AGC TECHNO GLASS CO., catalog number: SH90-20 )
  3. 50 ml culture tube (Thomas Scientific, Labcon, catalog number: 3181-345-008 )
  4. 1.5 ml microtube (BMbio, catalog number: BM-15 )
  5. Parafilm (VWR International, Bemis, catalog number: PM996 )
  6. Micropore surgical tape (3M, catalog number: 1530-0 )
  7. Qualitative filter paper No. 2 (Toyo Roshi Kaisha, Advantec, catalog number: 00021110 )
  8. Test tube for incubation of transformant (AGC TECHNO GLASS CO., catalog number: TST-SCR16-150 )
  9. pSA0104 Vector
  10. Species: Heterothallic C. psl. complex strains [NIES-67 (mt+) and NIES-68 (mt-) (the National Institute for Environmental Studies)]
  11. KOD-plus NEO DNA polymerase (TOYOBO CO., catalog number: KOD-401 )
  12. KOD-FX DNA polymerase (TOYOBO CO., catalog number: KFX-101 )
  13. GENEART seamless cloning and assembly kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A13288 )
  14. High Pure Plasmid Isolation kit (Roche Diagnostics, catalog number: 11754785001 )
  15. Spermidine (Wako Pure Chemical Industries, catalog number: 191-13831 )
  16. Agar powder (Nacalai tesque, catalog number: 01028-85 )
  17. Ethanol absolute (Wako Pure Chemical Industries, catalog number: 057-00451 )
  18. Glycerol (Wako Pure Chemical Industries, catalog number: 075-00616 )
  19. Quant-iT dsDNA Assay Kit, broad range (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q-33130 )
  20. Hygromycin B (Wako Pure Chemical Industries, catalog number: 085-06153 )
  21. QuickExtract Plant DNA Extraction Solution (Epicentre, catalog number: QEP80705 )
  22. Calcium nitrate tetrahydrate [Ca(NO3)2.4H2O] (Wako Pure Chemical Industries, catalog number: 039-00735 )
  23. Potassium nitrate (KNO3) (Wako Pure Chemical Industries, catalog number: 160-04035 )
  24. Disodium β-glycerophosphate pentahydrate (Sigma-Aldrich, catalog number: 50020-1000 G )
  25. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Nacalai tesque, catalog number: 21003-75 )
  26. Vitamin B12 (Wako Pure Chemical Industries, catalog number: 226-00343 )
  27. Biotin (Wako Pure Chemical Industries, catalog number: 023-08711 )
  28. Thiamine HCl (Wako Pure Chemical Industries, catalog number: 201-00852 )
  29. 2-amino-2-hydroxymethyl-1, 3-propanediol (Wako Pure Chemical Industries, catalog number: 011-16381 )
  30. Hydrochloric acid (HCl) (Nacalai tesque, catalog number: 18321-05 )
  31. Na2EDTA.2H2O (Wako Pure Chemical Industries, catalog number: 345-01865 )
  32. Iron(III) chloride hexahydrate (FeCl3.6H2O) (Wako Pure Chemical Industries, catalog number: 091-00872 )
  33. Manganese(II) chloride tetrahydrate (MnCl2.4H2O) (Nacalai tesque, catalog number: 21211-45 )
  34. Zinc sulfate heptahydrate (ZnSO4.7H2O) (Nacalai tesque, catalog number: 37011-62 )
  35. CoCl2.6H2O (Wako Pure Chemical Industries, catalog number: 003-00368 )
  36. Na2MoO4.2H2O (Wako Pure Chemical Industries, catalog number: 019-00247 )
  37. Calcium chloride dihydrate (CaCl2.2H2O) (Wako Pure Chemical Industries, catalog number: 039-00431 )
  38. C medium (see Recipes)
  39. PIV metals (see Recipes)
  40. Conditioned C medium (see Recipes)
  41. MI medium (see Recipes)

Equipment

  1. 300 ml Erlenmeyer flask (AGC TECHNO GLASS CO., catalog number: 4980FK300 )
  2. Growth chamber (Nippon Medical & Chemical Instruments, model: KCLP-1400II CT ), being discontinued
  3. Handmade hemocytometer (1 x 1 mm, grid length x grid width) (not commercially available)
  4. Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: veriti200)
  5. Centrifuge (Hitachi Ltd., model: CF16RX )
  6. Swing rotor (Hitachi Ltd., model: T5SS31 )
  7. Angle rotor (Hitachi Ltd., model: T15AP31 )
  8. Centrifuge (KUBOTA Corporation, model: 1920 ), being discontinued
  9. Angle rotor (KUBOTA Corporation, model: RA-48J ), being discontinued
  10. Cute mixer (EYELA, model: CM-1000 )
  11. Qubit fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32857 ), being discontinued
  12. Ultrasonic cleaner (Sigma-Aldrich, Branson®, model: 3510J-DTH )
  13. Gene transfer system (Tanaka co., model: IDERA GIE-III )
  14. Fluorescence stereomicroscope LEICA MZ16 F (Leica Microsystems)
  15. Micro Pick and Place System (Nepa Gene Co., catalog number: MPP-300 )
  16. Light microscope (Olympus, model: CK-40 ), being discontinued

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Abe, J., Hirano, N., Komiya, A., Kanda, N., Fujiwara, A., Hori, S., Tsuchikane, Y. and Sekimoto, H. (2016). Preparation of Knockdown Transformants of Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex. Bio-protocol 6(10): e1813. DOI: 10.21769/BioProtoc.1813.

ris ris Download Citation in RIS Format

分类

分子生物学 > DNA > 转化

植物科学 > 藻类学 > DNA > 转化

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件