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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   

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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), Alga(藻类), Closterium(新月藻), Charophycean(charophycean), Transformation(转型)

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

  1. Preparation of cells for transformation
    1. Culture vegetative cells in 150 ml C medium (http://www.nies.go.jp/biology/mcc/home.htm) at 23 °C under a 16 h light (28 µmol/m2/s) and 8 h dark cycle in 300 ml Erlenmeyer flasks.
    2. Collect the C. psl. complex cells from the mid-logarithmic to early stationary phase (9-11 days of culture) in a 50 ml culture tube and concentrate by centrifugation (1,100 x g for 2 min at 23 °C) using a swing-type rotor (T5SS31). For the slowdown, deceleration should be set to the slowest speed.
    3. Re-suspend the cells in a small aliquot of fresh C medium and count the cells under a light microscope (CK40) using a handmade hemocytometer. Adjust to a density of 5 x 106 cells ml-1 by adding fresh C medium.
    4. Spread the cell suspension (1 x 106 cells) using a glass bacterial spreader onto a 90-mm plate containing C medium with 1.5% agar (w/v), prepared one day previously and stored at room temperature.
    5. Culture the cells at 23 °C for 2 days under continuous light at 10 µmol m-2 s-1 (Figure 1).


      Figure 1. Photograph of gold particles sedimented by centrifugation

  2. Preparation of constructs for transformation of the C. psl. complex (approximately 4-5 days)
    1. To avoid off-target silencing, the DNA region used for the antisense expression should be carefully checked, using the BLASTN program, against unpublished RNAseq and a genome database of the C. psl. complex.
    2. Amplify the required region of target DNA by PCR with KOD-plus-NEO DNA polymerase from plasmid clones (2 min at 94 °C, followed by 30 cycles of 10 sec at 98 °C and 1 min/kbp at 68 °C). In the case of CpRLK1 gene, 2,180 bp fragment, encoding the extracellular domain of CpRLK1 protein, was amplified. The primer DNAs should include extra sequences corresponding to the vector sequences (ex. 5'-ccagcatgactagtctcgagTTCGGGCTGTTGCTTCGGCGTCA-3' and 5'-gcttcatcaaattactcgagTGGGTGCCGCCGTAGGTTAATAT-3'), which are required for cloning into the pSA0104 vector (Figure 2, Hirano et al., 2015) using the GENEART kit. The enzyme mix provided with the kit recognizes and assembles the vector and PCR fragments sharing terminal end-homology. The pSA0104 vector contains a promoter region of CpHSP70 (5’CpHSP70) for expression of the target gene and a hygromycin resistance gene (aph7”, Berthold et al., 2002) for selection (Figure 2).

      A

      B



      Figure 2. The plasmid vector for transformation of C. psl. complex. A. Constructs of pSA0104 and pSA0104_anti-GeneA. The promoters and untranslated regions of CpCAB1 and CpHSP70 genes are shown as light gray and white boxes, respectively. The aph7’’ gene (hygromycin resistance gene) is indicated by black box. Forward and reverse primers used for plasmid construction are shown as arrows under the boxes. Initiation codon (ATG) and stop codon (TAA) are indicated in their respective boxes. The plasmid backbone of the construct was pBluescript II SK+. K, KpnI; N, NotI; X, XhoI. B. The sequence of pSA0104. The restriction sites used for cloning and for linearization have been highlighted. Blue character indicates the promoter and untranslated regions of CpCAB1. Pink character indicates aph7’’ gene.

    3. Using the GENEART kit, insert the amplified DNA fragment corresponding to the antisense strand of a target gene into the XhoI site (immediately after pCpHSP70) of the pSA0104 vector.
    4. After confirmation of the sequence, prepare a sufficient amount of plasmid DNA from an E. coli culture using a High Pure Plasmid Isolation kit, or the standard Alkaline SDS method, followed by phenol/chloroform purification (Birnboim and Doly, 1979).
    5. Quantify the DNA concentration accurately using a Qubit fluorometer and the Quant-iT dsDNA Assay Kit.
    6. To linearize the constructs, digest the vector arm region using suitable restriction enzymes, according to the manufacturer’s instruction. In most cases, NotI, KpnI, or ScaI (on pBluescript II SK+) can be used (Figure 2).

  3. Microcarrier stock preparation
    1. Add 60 mg of gold particles and 1 ml of 70% (w/v) ethanol solution to a 1.5 ml microtube.
      Note: 0.25 µm of gold particles would result in a higher transformation efficacy than 0.6 µm (Abe et al., 2008), however, this may be expensive. Normally, 0.6 µm gold particles are used.
    2. Vortex at 2,000 rpm using a micromixer for 5 min.
    3. Allow to sediment for 15 min at room temperature.
    4. Further sediment by centrifugation using a T15AP31 rotor at 18,800 x g for 10 secfI.
    5. Carefully remove the solution with a pipette.
    6. Add 1 ml of sterile MilliQ water and vortex for 2 min. Sediment by centrifugation as in step B4. Discard the water. Repeat 3 times.
    7. Add 1 ml of 50% sterile glycerol solution and vortex for 1 min.
    8. Stored at -20 °C until needed.

  4. Coating the microcarriers with the plasmid DNA
    1. Thaw the microcarriers (step C8) and suspend by sonication for 5 min using an ultrasonic cleaner (set the mode “sonics”).
    2. Add the following reagents to the 82.5 µl (4.95 mg, for 20 shots) of microcarrier suspension in a 1.5 ml microtube. Mix by vortexing briefly after the addition of each reagent.
      1. 82.5 µl of linearized construct (for 400 ng/µl of empty pSA0104, which is 6,086 bp in length). Adjust the concentration to the equal mol value depending on the size of the construct.
      2. 250 µl of 2.5 M CaCl2 (sterile filtered)
      3. 100 µl of 0.1 M spermidine (sterile filtered)
    3. Vortex for 2 min.
    4. Incubate for 30 min at room temperature. Invert the tube gently every 10 min.
    5. Sediment by centrifugation at 8,300 x g for 10 sec (RA-48J rotor) using a Kubota 1920 centrifuge.
    6. Discard the supernatant without disturbing the microcarrier sediment.
    7. Add 750 µl of 70% (v/v) ethanol and vortex for 2 min.
    8. Sediment and discard the supernatant as in steps D5-6.
    9. Add 750 µl of absolute ethanol and vortex for 2 min.
    10. Sediment and discard the supernatant as in steps D5-6.
    11. Add 250 µl of absolute ethanol. Seal the cap with parafilm to minimize ethanol evaporation until needed.

  5. Particle bombardment
    Note: This protocol is optimized for the IDERA GIE-III gene transfer system (Figure 3). Further optimization may be required if other particle delivery systems are used.


    Figure 3. Photograph of IDERA GIE-III gene transfer system

    1. Prepare an autoclaved top agar solution [0.4% (w/v) agar in C medium] and incubate the melted agar solution in a water bath at 42 °C.
    2. Place a barotolerant chamber on a clean bench (Figure 4).


      Figure 4. Photograph of barotolerant chamber on a clean bench

    3. Suspend the microcarriers (step D11) completely by vortexing.
    4. Add 3.3 µl of microcarrier suspension into each of three wells (Figure 5) of autoclaved DNA cartridges.
      Note: Before adding the suspension, we recommend bombarding the empty cartridges to empty the dishes. This step cleans and prevents clogging of the wells.


      Figure 5. Photograph of DNA cartridge focusing on three wells. The arrows indicate the wells.

    5. Place the DNA cartridge and precultured C. psl. complex on the culture dish (step A5, Figure 6) into the chamber (Figure 7).


      Figure 6. Photograph of Closterium cells cultured for 2 days on 90-mm plate containing C medium with 1.5% agar (w/v)


      Figure 7. Photograph of barotoletant chamber just before the bombardment. Arrow indicates the DNA cartridge and arrowhead indicates the cultured plate.

    6. Start bombardment using the following parameters (Figure 8):
      Gun-to-target distance
      13.0 cm
      Helium exit pressure
      5.5 kgf/cm
      Vacuum in the barotolerant chamber
      710-715 mmHg
      Opening time of gas valve to allow rapid flow of helium gas
      0.05 sec


      Figure 8. Photograph of IDERA GIE-III focusing on the parameters

  6. Selection of hygromycin resistant cells
    1. Immediately after bombardment, add 5 ml of melted top agar (step E1) and allow to stand for 30 min.
    2. Add 4 ml of C medium to the agar plate. Seal the plate using surgical tape and incubate for 2 d at 23 °C under continuous light (10 µmol m-2 s-1) to enable accumulation of the aminoglycoside phosphotransferase (aph’’ protein).
    3. Carefully discard the C medium on the agar by decantation. Do not disturb the top surface of the agar.
    4. Add 5 ml of C medium containing 50-100 µg ml -1 hygromycin B to the plate to select the hygromycin-resistant colonies.
      Note: The suitable concentration of hygromycin B for screening should be tested before transformation as this depends on the strains used. In our experience, a lower concentration (10 µg ml-1) is suitable to select for certain C. psl. complex strains.
    5. Incubate the cells for 3 to 5 weeks, with a weekly replacement of fresh. hygromycin-containing medium, under continuous light at 23 °C.
    6. Pick the surviving colonies (Figure 9) and streak onto 0.8% (w/v) agar plates containing conditioned C medium (Abe et al., 2011) and hygromycin. The unknown factor(s) for cell proliferation, which was secreted from growing cells into surrounded environment, would be included in the conditioned medium and would facilitate the cell division.
      Note: Use a stereomicroscope to check for contamination of the colonies with bacteria and/or fungi (Figure 10). If contamination occurs, wash the colony repeatedly (at least three times) by the capillary washing method (Andersen and Kawachi, 2005) or isolate a single cell using the Micro Pick and Place System.


      Figure 9. Photograph of surviving colonies after the selection. Arrows indicate the survived colonies, which have been incubated in C medium containing 50 µg ml-1 hygromycin B for 5 weeks.


      Figure 10. Photograph of a contaminated colony. Scale bar = 200 µM

    7. Pick the single colony on the plates (step F6) and transfer to 5 ml of conditioned medium in a test tube. Incubate at 23 °C under a 16 h light and 8 h dark cycle for 2-3 weeks.
      Note: If contamination occurs, washing and isolation of a single cell is required as described in Note of step F6.
    8. Transfer the proliferated cells from step F7 to fresh C medium and maintain under normal conditions (step A1).

  7. Confirmation of successful integration of the constructs into the genome
    1. Isolate crude genomic DNA from cells using QuickExtract Plant Extraction Solution, according to the manufacturer’s instruction.
    2. Amplify the integrated DNA from the genomic DNA by PCR with KOD FX DNA polymerase (2 min at 94 °C, followed by 40 cycles of 15 sec at 98 °C and 1 min/kbp at 68 °C).

  8. Evaluation of knockdown effects
    1. If specific antibody against the target protein is being used, check the protein expression levels by Western blotting.
    2. Select those transformants showing a high reduction in protein expression levels for further characterization, because the knockdown effect will vary and some will not show a distinct reduction in protein expression.
    3. Evaluate the phenotype of the selected knockdown transformants. In our case, the sexual responses of transformants in MI medium was mostly evaluated.

Recipes

  1. C medium
    Stock solution for 5 L of C media
    10% (w/v) Ca(NO3)2.4H2O solution
    7.5 ml
    5% (w/v) KNO3 solution
    10 ml
    5% (w/v) disodium β-glycerophosphate pentahydrate solution
    5 ml
    4% (w/v) MgSO4.7H2O solution
    5 ml
    0.0001% (w/v) vitamin B12 solution
    0.5 ml
    0.0001% (w/v) biotin solution
    0.5 ml
    0.0112% (w/v) thiamine HCl solution
    0.45 ml
    PIV metals
    15 ml
    2-amino-2-hydroxymethyl-1, 3-propanediol
    2.5 g
    Adjust pH to 7.5 with HCl
    a. PIV metals
    Component (for 500 ml)
    Na2EDTA.2H2O
    500 mg
    FeCl3.6H2O
    98 mg
    MnCl2.4H2O
    18 mg
    ZnSO4.7H2O
    11 mg
    CoCl2.6H2O
    2 mg
    Na2MoO4.2H2O
    1.25 mg
  2. Conditioned C medium (Abe et al., 2011)
    Incubate wild-type mt+ or mt- cells in fresh C medium for 14-20 days
    Collect the cultured medium by filtration using qualitative filter paper and sterilize the filtered medium by autoclaving (121 °C for 15 min)
  3. MI medium
    Stock solution for 5 L of MI media
    5% (w/v) CaCl2.2H2O solution
    10 ml
    5% (w/v) disodium β-glycerophosphate pentahydrate solution
    5 ml
    4% (w/v) MgSO4.7H2O solution
    5 ml
    0.0001% (w/v) vitamin B12 solution
    0.5 ml
    0.0001% (w/v) biotin solution
    0.5 ml
    0.0112% (w/v) thiamine HCl solution
    0.45 ml
    PIV metals
    15 ml
    2-amino-2-hydroxymethyl-1, 3-propanediol
    2.5 g
    Adjust pH to 8.5 with HCl

Acknowledgments

The authors wish to thank Dr. Kensuke Ichihara (Univ. Tokyo) and Ms. Wakana Takiguchi (JWU) for their technical supports. This work was partly supported by Grants-in-Aid for Scientific Research (nos. 24370038, 24247042, 25304012, 26650147, 15H05237 to H.S., no. 23770277 to J. A., nos. 23770093 and 26440223 to Y. T.) from the Japan Society for the Promotion of Science, Japan, a grant from the New Technology Development Foundation to H. S. and Y. T., and MEXT-supported Program for the Strategic Research Foundation at Private Universities to H. S.

References

  1. Abe, J., Hiwatashi, Y., Ito, M., Hasebe, M. and Sekimoto, H. (2008). Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex. Plant Cell Physiol 49(4): 625-632.
  2. Abe, J., Hori, S., Tsuchikane, Y., Kitao, N., Kato, M. and Sekimoto, H. (2011). Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex. Plant Cell Physiol 52(9): 1676-1685.
  3. Andersen, R. A. and Kawachi, M. (2005) Chapter 6: Traditional microalgae isolation techniques, In: Andersen R. A. (ed). Algal Culturing techniques. Elsevier Academic Press, pp 83-100 (total 578 pages).
  4. Berthold, P., Schmitt, R. and Mages, W. (2002). An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153(4): 401-412.
  5. Birnboim, H. C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7(6): 1513-1523.
  6. Hirano, N., Marukawa, Y., Abe, J., Hashiba, S., Ichikawa, M., Tanabe, Y., Ito, M., Nishii, I., Tsuchikane, Y. and Sekimoto, H. (2015). A receptor-like kinase, related to cell wall sensor of higher plants, is required for sexual reproduction in the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex. Plant Cell Physiol 56(7): 1456-1462.

简介

为了制备鬼臼藓藻(Clonsterium peracerosum-strigosum-littorale)复合物的敲低转化体,用新构建的载体(pSA0104)应用粒子轰击,所述载体具有内源性 组成型启动子,其与对应于靶基因的反义链的DNA片段融合。 使用潮霉素抗性基因( aph7")选择潮霉素抗性菌落。 在第二次筛选后,通过PCR检查载体到基因组中的整合,并使用针对靶蛋白的特异性抗体通过蛋白质印迹评估敲低效应。

关键字:击倒, 藻类, 新月藻, charophycean, 转型

材料和试剂

  1. 0.6微米金微载体(Bio-Rad Laboratories,目录号:165-2262)或0.25微米金纳米颗粒(BBI Solutions,目录号:EMGC250)
  2. 细胞培养皿,90mm×20mm聚苯乙烯(AGC TECHNO GLASS CO。,目录号:SH90-20)
  3. 50ml培养管(Thomas Scientific,Labcon,目录号:3181-345-008)
  4. 1.5ml微量管(BMbio,目录号:BM-15)
  5. Parafilm(VWR International,Bemis,目录号:PM996)
  6. 微孔手术胶带(3M,目录号:1530-0)
  7. 定性滤纸No.2(Toyo Roshi Kaisha,Advantec,目录号:00021110)
  8. 转化体培养用试管(AGC TECHNO GLASS CO。,目录号:TST-SCR16-150)
  9. pSA0104矢量
  10. 物种:Heterothallic c。 复合菌株[NIES-67(mt + )和NIES-68(mt - )(国家环境研究所) >
  11. KOD-plus NEO DNA聚合酶(TOYOBO CO。,目录号:KOD-401)
  12. KOD-FX DNA聚合酶(TOYOBO CO。,目录号:KFX-101)
  13. GENEART无缝克隆和装配试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:A13288)
  14. 高纯质粒分离试剂盒(Roche Diagnostics,目录号:11754785001)
  15. 亚精胺(Wako Pure Chemical Industries,目录号:191-13831)
  16. 琼脂粉(Nacalai tesque,目录号:01028-85)
  17. 乙醇绝对(Wako Pure Chemical Industries,目录号:057-00451)
  18. 甘油(Wako Pure Chemical Industries,目录号:075-00616)
  19. Quant-iT dsDNA测定试剂盒(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q-33130)
  20. 潮霉素B(Wako Pure Chemical Industries,目录号:085-06153)
  21. QuickExtract植物DNA提取溶液(Epicentre,目录号:QEP80705)
  22. 四水合硝酸钙[Ca(NO 3)2 sub] 4H 4H 2 O](Wako Pure Chemical Industries,目录 number:039-00735)
  23. 硝酸钾(KNO 3)(Wako Pure Chemical Industries,目录号:160-04035)
  24. β-磷酸甘油五水合物二钠(Sigma-Aldrich,目录号:50020-1000G)
  25. 硫酸镁七水合物(MgSO 4·7H 2 O 7H 2 O)(Nacalai tesque,目录号:21003-75)
  26. 维生素B 12(Wako Pure Chemical Industries,目录号:226-00343)
  27. 生物素(Wako Pure Chemical Industries,目录号:023-08711)
  28. 硫胺HCl(Wako Pure Chemical Industries,目录号:201-00852)
  29. 2-氨基-2-羟甲基-1,3-丙二醇(Wako Pure Chemical Industries,目录号:011-16381)
  30. 盐酸(HCl)(Nacalai tesque,目录号:18321-05)
  31. (和光纯药工业公司,目录号:345-01865)。< br /></sup>
  32. 氯化铁(III)六水合物(FeCl 3·6H 2 O)6H 2 O)(Wako Pure Chemical Industries,目录号:091-00872)
  33. (Nacalai tesque,目录号:21211-45)的氯化锰(II)四水合物(MnCl 2 4H 2 O)
  34. 硫酸锌七水合物(ZnSO 4·7H 2 O·7H 2 O)(Nacalai tesque,目录号:37011-62)
  35. (Wako Pure Chemical Industries,目录号:003-00368)。
  36. (Wako Pure Chemical Industries,目录号:019-00247)的Na 2 SO 4,Na 2 SO 4,Na 2 SO 4, br />
  37. 氯化钙二水合物(CaCl 2·2H 2·2H 2 O)(Wako Pure Chemical Industries,目录号:039-00431)
  38. C介质(见配方)
  39. PIV金属(参见配方)
  40. 条件C培养基(见配方)
  41. MI培养基(见配方)

设备

  1. 300毫升锥形烧瓶(AGC TECHNO GLASS CO。,目录号:4980FK300)
  2. 生长室(Nippon Medical& Chemical Instruments,型号:KCLP-1400II CT),中断
  3. 手工血球计(1 x 1 mm,网格长度x网格宽度)(不可商购)
  4. 热循环仪(Thermo Fisher Scientific,Applied Biosystems TM ,型号:veriti200)
  5. 离心机(Hitachi Ltd.,型号:CF16RX)
  6. 摆动转子(Hitachi Ltd.,型号:T5SS31)
  7. 角转子(Hitachi Ltd.,型号:T15AP31)
  8. 离心机(久保田公司,型号:1920),中断了
  9. 角转子(久保田公司,型号:RA-48J),中断了
  10. 可爱的调音台(EYELA,型号:CM-1000)
  11. Qubit荧光计(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q32857),被中断
  12. 超声清洁剂(Sigma-Aldrich,Branson ,型号:3510J-DTH)
  13. 基因转移系统(田中公司,型号:IDERA GIE-III)
  14. 荧光立体显微镜LEICA MZ16 F(Leica Microsystems)
  15. Micro Pick and Place System(Nepa Gene Co.,目录号:MPP-300)
  16. 光显微镜(Olympus,型号:CK-40),中断了

程序

  1. 转化用细胞的制备
    1. 在150ml C培养基中培养营养细胞( http://www.nies.go.jp/biology/mcc/home。 htm )在23℃下在16小时光照下   (28μmol/m 2 s/s)和8小时暗循环在300ml锥形瓶中。
    2. 收集C. psl。 复杂细胞从中对数到早 固定相(培养9-11天)在50ml培养管中 通过离心浓缩(1,100×g,在23℃下2分钟),使用a 摆动式转子(T5SS31)。 对于减速,应设置减速度 到最慢的速度。
    3. 重悬细胞在一小部分   新鲜C培养基并在光学显微镜下计数细胞(CK40) 使用手工血细胞计数器。 调节至5×10 6个细胞的密度 ml -1
    4. 传播细胞悬液(1 x 10 细胞)使用玻璃细菌涂布器涂布在90mm板上含有1.5%琼脂(w/v)的C培养基,一天前制备 并在室温下储存
    5. 在23℃下在10μmolm -2 s -1 -1的连续光下培养细胞2天(图1)。


      图1.通过离心沉淀的金颗粒的照片

  2. 制备用于转化C的构建体。 psl。复杂(约4-5天)
    1. 为了避免脱靶沉默,用于反义的DNA区域 表达应仔细检查,使用BLASTN程序, 针对未发表的RNAseq和C的基因组数据库。 psl。复杂
    2. 用KOD-plus-NEO通过PCR扩增靶DNA的所需区域  DNA聚合酶(在94℃2分钟,然后30分钟) 98℃下10秒和68℃下1分钟/kbp的循环)。在 CpRLK1 基因的情况下,编码CpRLK1的细胞外结构域的2180bp片段 蛋白质。引物DNA应包括额外的序列 对应于载体序列 5'-ccagcatgactagtctcgagTTCGGGCTGTTGCTTCGGCGTCA-3'和 5'-gcttcatcaaattactcgagTGGGTGCCGCCGTAGGTTAATAT-3'),这是必需的 用于克隆到pSA0104载体中(图2,Hirano等人,2015) 使用GENEART试剂盒。试剂盒提供的酶混合物识别 并组装载体和PCR片段共享终端 末端同源性。 pSA0104载体含有用于靶基因表达的CpHSP70( 5'CpHSP70 )的启动子区域和潮霉素 抗性基因( aph7",Berthold等人,2002)进行选择(图2)。
      A

      B



      图2.用于转化C的质粒载体。 psl。复杂。 pSA0104和pSA0104_anti- GeneA的构建体。启动子和 CpCAB1 和 CpHSP70 基因的非翻译区显示为浅灰色  和白盒子。 aph7''基因(潮霉素抗性 基因)用黑框表示。正向和反向引物用于 质粒构建如框下方的箭头所示。引发 密码子(ATG)和终止密码子(TAA) 框。构建体的质粒主链是pBluescript II SK +。 , N , NotI ; X , XhoI 。 B.pSA0104的序列。限制 已经突出了用于克隆和线性化的位点。 蓝色字符表示CpCAB1的启动子和非翻译区。粉红色字符表示 aph7''基因。

    3. 使用 GENEART试剂盒,插入扩增的DNA片段对应的 靶基因的反义链插入pSA0104载体的 Xho I位点(紧接在pCpHSP70 之后)。
    4. 确认后 序列,从E制备足够量的质粒DNA。 使用高纯质粒分离试剂盒或标准品的大肠杆菌培养物 碱性SDS法,随后苯酚/氯仿纯化 (Birnboim和Doly,1979)
    5. 使用Qubit荧光计和Quant-iT dsDNA测定试剂盒精确定量DNA浓度
    6. 为了线性化构建体,使用消化载体臂区 合适的限制酶,根据制造商的 指令。 在大多数情况下,不是我, Kpn 我或 sca 我(在pBluescript II SK + 可以使用(图2)。

  3. 微载体原液制备
    1. 向1.5ml微量管中加入60mg金颗粒和1ml 70%(w/v)乙醇溶液。
      注意:0.25μm的金颗粒将导致更高的转变   功效比0.6μm(Abe等人,2008),然而,这可能是 昂贵。 通常使用0.6μm的金颗粒。
    2. 使用微混合器在2,000rpm下涡旋5分钟
    3. 允许在室温下沉淀15分钟。
    4. 通过使用T18AP31转子在18,800×g下离心10秒进一步沉淀。
    5. 用移液管小心取出溶液。
    6. 加入1ml无菌MilliQ水并涡旋2分钟。 沉积物 如步骤B4中那样离心。 丢弃水。 重复3次。
    7. 加入1ml 50%无菌甘油溶液并涡旋1分钟
    8. 储存于-20°C直至需要

  4. 用质粒DNA包被微载体
    1. 解冻微载体(步骤C8),并使用超声波清洗机(设置模式"sonics")通过超声处理5分钟悬浮。
    2. 将以下试剂加入82.5μl(4.95mg,20次)   微载体悬浮液在1.5ml微管中。 通过短暂涡旋混合   加入每种试剂后。
      1. 82.5μl线性化 构建体(对于400ng /μl的空pSA0104,其长度为6,086bp)。   根据尺寸将浓度调节至相等的摩尔值 的结构。
      2. 250μl2.5M CaCl 2(无菌过滤)
      3. 100μl的0.1M亚精胺(无菌过滤)
    3. 涡旋2分钟。
    4. 在室温下孵育30分钟。 每10分钟轻轻颠倒管。
    5. 使用Kubota 1920离心机以8,300×g离心10秒(RA-48J转子)沉淀沉淀物。
    6. 丢弃上清液,不影响微载体沉淀
    7. 加入750μl的70%(v/v)乙醇并涡旋2分钟
    8. 沉淀并弃去上清液,如步骤D5-6。
    9. 加入750μl无水乙醇并涡旋2分钟
    10. 沉淀并弃去上清液,如步骤D5-6。
    11. 加入250μl无水乙醇。 用石蜡膜密封帽以最小化乙醇蒸发直到需要。

  5. 粒子轰击
    注意:该方案针对IDERA GIE-III基因转移系统进行了优化(图3)。 如果使用其他颗粒输送系统,可能需要进一步优化

    图3. IDERA GIE-III基因转移系统的照片

    1. 准备高压灭菌顶部琼脂溶液[0.4%(w/v)琼脂的C培养基] 并在42℃的水浴中孵育熔化的琼脂溶液。
    2. 将一个耐压室放在一个干净的工作台上(图4)

      图4.清洁工作台上的耐压室照片

    3. 通过涡旋使微载体完全悬浮(步骤D11)
    4. 在高压灭菌的DNA试剂盒的三个孔(图5)的每一个中加入3.3μl微载体悬浮液 注意:在添加暂停之前,我们建议您轰炸空 墨盒清空盘子。 此步骤清洁并防止堵塞的井。


      图5.集中在三个孔上的DNA盒的照片。 箭头表示油井。

    5. 放置DNA试剂盒并预培养。 在培养皿(步骤A5,图6)上进入培养皿(图7)。

      图6.在含有含1.5%琼脂(w/v)的C培养基的90-mm平板上培养2天的Closterium细胞的照片。


      图7.正好在baro托管室前的照片 箭头表示DNA弹夹,箭头指示 培养板
    6. 使用以下参数开始轰击(图8):
      枪至目标距离
      13.0厘米
      氦出口压力
      5.5 kgf/cm 2
      在耐压容器中真空
      710-715 mmHg
      气阀打开时间允许氦气快速流动
      0.05秒


      图8.关注参数的IDERA GIE-III的照片

  6. 选择潮霉素抗性细胞
    1. 轰击后立即加入5ml熔化的顶层琼脂(步骤E1),并放置30分钟。
    2. 加入4毫升C培养基的琼脂板。 使用密封板 并在23℃下在连续光下孵育2天 μmolm -2 -2 s -1 sup -1),以使得氨基糖苷类 磷酸转移酶(aph"蛋白)
    3. 通过倾析小心地丢弃琼脂上的C培养基。 不要打扰琼脂的顶部表面。
    4. 向平板中加入5ml含有50-100μg/ml抗潮霉素B的C培养基,以选择潮霉素抗性菌落。
      注意:用于筛选的潮霉素B的合适浓度 在转化之前测试,因为这取决于使用的菌株。 在 我们的经验,较低的浓度(10μg/ml )适合选择   对于某些C.psl。 复杂菌株。
    5. 孵育细胞3 至5周,每周更换新鲜。 含潮霉素 介质,在23℃的连续光照下
    6. 选择幸存的 菌落(图9),并且在含有0.8%(w/v)琼脂平板上划线 条件C培养基(Abe等人,2011)和潮霉素。 未知 细胞增殖的因子,其由生长的细胞分泌进入包围的环境中,将包括在条件培养基中  并且将促进细胞分裂 注意:使用 立体显微镜检查菌落的污染 细菌和/或真菌(图10)。如果发生污染,请清洗 通过毛细管洗涤法重复(至少三次)菌落  (Andersen和Kawachi,2005)或使用Micro分离单个细胞 选择和放置系统。


      图9.存活菌落的照片 箭头表示存活的菌落 在含有50μg/ml潮霉素B的C培养基中温育5 周。


      图10.受污染菌落的照片。 比例尺= 200μM

    7. 选择平板上的单个菌落(步骤F6),并转移至5ml 的条件培养基。 在23℃下孵育16小时 光和8小时黑暗周期2-3周 注意:如果发生污染,则需要按照步骤F6的注释中描述的 清洗和隔离单个单元。
    8. 将来自步骤F7的增殖细胞转移到新鲜C培养基中并在正常条件下保持(步骤A1)。

  7. 确认构建体成功整合到基因组中
    1. 根据制造商的说明,使用QuickExtract Plant Extraction Solution从细胞中分离粗基因组DNA
    2. 通过PCR用KOD FX从基因组DNA扩增整合的DNA DNA聚合酶(94℃2分钟,然后94℃15秒,98℃)   和68℃下1分钟/kbp)。

  8. 击倒效应的评价
    1. 如果正在使用针对靶蛋白的特异性抗体,则通过Western印迹检查蛋白表达水平。
    2. 选择那些显示蛋白质高度降低的转化体 表达水平进一步表征,因为敲除 效果将不同,并且一些不会显示蛋白质的明显减少 表达式
    3. 评估所选基因敲低的表型 转化体。 在我们的情况下,转化子在心肌梗死的性反应 大多数评价培养基。

食谱

  1. C介质
    5升C介质的储存溶液
    10%(w/v)Ca(NO 3)2 sub缓冲溶液
    7.5 ml
    5%(w/v)KNO 3溶液
    10 ml
    5%(w/v)β-甘油磷酸五钠水溶液
    5 ml
    4%(w/v)MgSO 4溶液
    5 ml
    0.0001%(w/v)维生素B 12溶液
    0.5 ml
    0.0001%(w/v)生物素溶液 0.5 ml
    0.0112%(w/v)硫胺素HCl溶液 0.45 ml
    PIV金属
    15 ml
    2-氨基-2-羟甲基-1,3-丙二醇
    2.5克
    用HCl
    调节pH至7.5 一个。 PIV金属
    组分(500ml)
    Na EDTA 2H 2 500毫克
    FeCl 3 6H <2> O
    98 mg
    MnCl 2 4H O
    18 mg
    ZnSO 4 。 7H O
    11 mg
    COCl <2> 6H <2> O
    2 mg
    Na 2 MoO 4 sub 。 2H 2
    1.25 mg
  2. 条件C培养基(Abe et al。,2011)
    在新鲜C培养基中孵育野生型mt + 或mt - 细胞14-20天
    通过使用定性滤纸过滤收集培养基并通过高压灭菌(121℃15分钟)对过滤的培养基灭菌
  3. MI介质
    5升MI培养基的库存溶液
    5%(w/v)CaCl 2溶液。 2H 2 O溶液
    10 ml
    5%(w/v)β-甘油磷酸五钠水溶液
    5 ml
    4%(w/v)MgSO 4溶液
    5 ml
    0.0001%(w/v)维生素B 12溶液
    0.5 ml
    0.0001%(w/v)生物素溶液 0.5 ml
    0.0112%(w/v)硫胺素HCl溶液 0.45 ml
    PIV金属
    15 ml
    2-氨基-2-羟甲基-1,3-丙二醇
    2.5克
    用HCl
    调节pH至8.5

致谢

作者感谢Kensuke Ichihara博士(东京大学)和Wakana Takiguchi女士(JWU)的技术支持。这项工作得到了日本促进协会的部分支持(科学研究助学金(No。)24370038,24247042,25304012,26650147,15H05237到HS,No.23770277到JA,No.23770093和26440223到YT)日本科学基金会,新技术开发基金会给HS和YT的资助,以及MEXT支持的私立大学战略研究基金项目

参考文献

  1. Abe,J.,Hiwatashi,Y.,Ito,M.,Hasebe,M。和Sekimoto,H。(2008)。 在内源性HSP70和CAB启动子控制下的外源基因在Cloacerium peracerosum-strigosum-littorale中的表达复合物。 植物细胞生理学 49(4):625-632
  2. Abe,J.,Hori,S.,Tsuchikane,Y.,Kitao,N.,Kato,M。和Sekimoto,H。 稳定的核转变的Closterium peracerosum-strigosum-littorale复杂。植物Cell Physiol 52(9):1676-1685。
  3. Andersen,R.A。和Kawachi,M。(2005)第6章:Traditional microalgae isolation techniques,In:Andersen R.A。(ed)。 <藻类培养技术。 Elsevier Academic Press,pp 83-100(共578页)。
  4. Berthold,P.,Schmitt,R。和Mages,W。(2002)。 工程化的链霉菌属 hygroscopicu s aph 7 "基因介导对莱茵衣藻中潮霉素B的显性抗性。 153(4):401-412。
  5. Birnboim,H.C.and Doly,J。(1979)。 用于筛选重组质粒DNA的快速碱性提取程序核酸研究 7(6):1513-1523
  6. Hirano,N。,Marukawa,Y.,Abe,J.,Hashiba,S.,Ichikawa,M.,Tanabe,Y.,Ito,M.,Nishii,I.,Tsuchikane,Y.and Sekimoto, 2015)。 与高等植物的细胞壁传感器相关的受体样激酶是有性生殖需要的植物细胞生理学56(7):1456-1462。单核细胞藻类
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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.
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