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Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection
DNA显微注射法生成秀丽隐杆线虫转基因动物   

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Abstract

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.

Keywords: Caenorhabditis elegans(秀丽隐杆线虫), Genetic transformation(遗传转化), Microinjection(显微注射), Extrachromosomal arrays(染色体外阵列), Germline(生殖细胞系), GFP(GFP), Transgenic animals(转基因动物)

Background

In C. elegans, DNA transformation by microinjection is commonly used to produce transgenic animals that over-express or ectopically express genes, which can be fused to tags (e.g., green fluorescent protein [GFP]), allowing for the phenotypic rescue of mutants, and/or the analysis of localization and function of proteins (Carter et al., 1990; Chalfie et al., 1994; Mello and Fire, 1995). The advent of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system requires microinjection to achieve highly specific genome editing by introducing point mutations or insertion/deletion mutations (summarized in Dickinson and Goldstein, 2016). Further, the technique is applied for the inducible and/or tissue-specific in vivo transcription of dsRNA to facilitate heritable RNA interference (RNAi) (Tavernarakis et al., 2000). The genetic material is injected into the distal gonad syncytium of adult hermaphrodite animals where it enters the developing oocytes (Figure 1). The exogenous DNA is arranged into large extrachromosomal arrays consisting of 50 to 300 copies, which are inherited in a non-Mendelian fashion to the following generations. Next-generation progeny (F1) carry the extrachromosomal array with a varying probability (between 5 to 80% [Stinchcomb et al., 1985; Mello et al., 1991]). Transgenic animals are identified amongst the offspring by the use of co-transformation markers, which are injected along with the DNA of interest (Table 1). Most commonly used are the pharyngeal expression of green fluorescent protein (GFP) or red fluorescence (mCherry), or the dominant rol-6(su1006) allele, which induces a distinct rolling phenotype in transgenic F1 progeny (Mello et al., 1991; Tabara et al., 1996; Frokjaer-Jensen et al., 2008).

Table 1. Commonly used co-transformation markers with distinct phenotypes for selection of transgenic animals upon microinjection in C. elegans



Figure 1. Microinjection scheme for C. elegans. A. Scheme of an adult C. elegans displaying the major organs including the pharynx, intestine and the gonad. When microinjecting C. elegans the injection capillary needs to be inserted in the syncytium (cytoplasmic core) of the distal gonad. The inlay indicates the area of interest for injection. B. DIC image of the area indicated in 1A. The nuclei of the germ-cells are clearly visible and surround the syncytium. Size bar corresponds to 50 µm.

Materials and Reagents

  1. Sterile pipette tips
  2. Microloader pipette tips (Eppendorf, catalog number: 5242956003 )
  3. Microinjection capillaries (Eppendorf Femtotips II, 0.5 µm inner and 0.7 µm outer diameter) (Eppendorf, catalog number: 930000043 )
  4. Microscope slides, 76 x 26 mm (Carl Roth, catalog number: 0656.1 )
  5. Microscope cover glasses, 24 x 60 mm (VWR, catalog number: 631-1575 )
  6. Tape (~1 mm thickness)
  7. Greiner Petri dishes (60 x 15 mm) (Sigma-Aldrich, catalog number: P5237 )
  8. Glass Pasteur Pipettes, disposable, 150 mm (BRAND, catalog number: 747715 )
  9. C. elegans animals (e.g., available from Caenorhabditis Genetics Center [CGC], University of Minnesota, MN, USA)
  10. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  11. DNA of interest for injection (plasmid, fosmid, PCR fragment, or similar) and plasmid containing a co-transformation marker (see Table 1)
  12. Miniprep Kit for DNA purification (e.g., QIAGEN-tip 20) (QIAGEN, catalog number: 10023 )
  13. Halocarbon oil 700 (Sigma-Aldrich, catalog number: H8898-100ML )
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  15. Peptone (BD, BactoTM, catalog number: 211677 )
  16. Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S6501 )
  17. Agar (Sigma-Aldrich, catalog number: 05040 )
  18. 100% ethanol
  19. Calcium chloride dehydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  20. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  21. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
  22. Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
  23. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: P018.1 )
  24. Di-Sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: T876.1 )
  25. Di-Potassium hydrogen phosphate (K2HPO4) (Carl Roth, catalog number: 5066.1 )
  26. Agarose (Biozym Scientific, catalog number: 840004 )
  27. OP50 seeded NGM agar plates (see Recipes)
  28. 2% agarose pads (see Recipes)
  29. Hairpin pick (see Recipes)
  30. M9 buffer (see Recipes)

Equipment

  1. Bunsen burner
  2. Cylindrical glass beaker
  3. Wormpick with platinum wire and pre-flattened tip (Genesee Scientific, catalog numbers: 59-AWP and 59-30P6 )
  4. Microwave
  5. Heat plate (e.g., IKA, model: C-MAG HP 4 , catalog number: 0003581600)
  6. Puller (optional; e.g., Sutter Instrument, model: P-97 )
  7. Autoclave
  8. Stereomicroscope (e.g., Leica Microsystems, model: Leica M80 ), optionally fluorescent stereomicroscope (e.g., Leica Microsystems, model: Leica M165 FC)
  9. Microscope for Microinjection (ZEISS, model: Axio Observer.A1 equipped with differential interference contrast [DIC] prisms, gliding table, objective 10x/0.3 M27, objective 40x/0.75 M27, optionally camera AxioCam ERc 5 sec and monitor for observation and demonstration purposes)
  10. Micromanipulator (e.g., Eppendorf InjectMan 4) (Eppendorf, catalog number: 5192000019 )
  11. Microinjector unit (e.g., Eppendorf FemtoJet 4i) (Eppendorf, catalog number: 5252000013 )
  12. Microcentrifuge (e.g., Eppendorf Centrifuge 5424) (Eppendorf, model: 5424 , catalog number: 5404000014)
  13. Spectrophotometer (e.g., Thermo Fisher Scientific, model: NanoDropTM 8000 , catalog number: ND-8000-GL)

Procedure

  1. Preparing the DNA and the injection unit
    1. Follow the instructions of the QIAGEN-tip 20 miniprep kit for DNA purification (see Notes).
    2. At the final step, elute the DNA in distilled H2O and determine the concentration with a spectrophotometer.
    3. Prepare a mix of 50 ng/µl of the DNA of interest and the co-transformation marker each (see Table 1) in a 100 µl volume.
    4. Centrifuge the solution in a microcentrifuge at maximum speed for 15 min, preferably at 4 °C. This step is necessary to precipitate floating particles in the DNA solution to prevent clogging of the microinjection capillary.
    5. Pipette 5 µl from top of the DNA solution to a new reaction tube and directly use it for microinjection. Any left-over can be frozen at -20 °C for later use.
    6. A few minutes prior to the injection procedure, switch on the microscope for microinjection, the microinjector unit and the micromanipulator (overview in Figure 2). Follow the instructions of the microinjector unit to pressurize and adjust the system. Set the ‘inject’ function to release a volume of approx. 10 picoliter (pl).


      Figure 2. Basic components of a setup for C. elegans microinjection. A. Overview over the setup and all its components: (a) light microscope with a 10x and a 40x objective and DIC optics, (b) microinjector unit for pressure appliance, (c1) micromanipulator control for all three dimensions (x, y, z axis) with fine and coarse control, (c2) micromanipulator-controlled capillary (needle) holder, (d) gliding table to flexibly move immobilized animals into the needle, (e) stereomicroscope for mounting the animals on agarose pads and recovery after injection, (f) optionally a monitor, connected to a camera for recording and demonstration purposes, (g) glass capillary (injection needle). B. Detailed view on the gliding table (d), the needle holder (c2) and the needle (g) attached. C. Close-up on the injection needle (Femtotips II, Eppendorf), which can be filled by microloader pipette tips.

    7. Use the microloader pipette tips to pipette 1 µl of the clean DNA solution into a microinjection capillary (see Notes). This volume is enough to inject more than a hundred animals.
    8. Carefully remove the cover of the capillary and screw it into the needle holder of the micromanipulator. The capillary force will collect the solution in the tip of the needle.

  2. Preparing the animals for injection
    1. One day prior to the injection procedure, use a stereoscope and a wormpick to transfer 40 L4 stage animals from a mixed population to freshly OP50-seeded NGM agar plates (see Recipes).
    2. Grow the animals at 20 °C overnight into young gravid adults with a fully developed germline (Figure 1A).
    3. One hour prior to the injection procedure shift the animals to 15 °C. This reduces the agility of animals, making them easier to handle for the injection.
    4. Place a drop of about 50 µl halocarbon oil on a 2% agarose pad (see Recipes) next to the agarose area. Under the stereoscope, transfer 10-20 worms into the oil (dipping the worm pick into the oil makes it easier to transfer the worms). Let the animals move in the oil for 5-10 min to remove bacteria that are attached to their cuticle as bacterial remnants can quickly clog the microinjection capillary.
    5. Place another small drop of about 20 µl halocarbon oil on the surface of the agar pad.
    6. Use the hairpin pick (see Recipes) to transfer one animal from the halocarbon oil next to the agarose into the drop on the surface of the agarose pad.
    7. Use the maximum magnification of the stereoscope to gently press the animal to the agarose surface with the hairpin pick. The animals will attach to the dry agarose surface (Figure 3).
    8. Place the agarose pad on the gliding table of the microscope for microinjection and swiftly commence with the injection procedure, as the animals will desiccate within approx. 10-15 min.


      Figure 3. An animal immobilized on a 2% agarose pad and ready for injection. The arrows indicate the preferred sites for injection in the gonad. Scale bar is 50 µm.

  3. Injection procedure
    1. Use the 10x objective to localize the immobilized animal in the halocarbon oil drop on the agarose pad.
    2. Use the micromanipulator in ‘coarse’ mode to move the tip of the injection capillary along the x-y axis in close proximity of the worm and then use the ‘fine’ mode of the micromanipulator z-axis control to roughly bring the tip of the needle in focus (Video 1).
    3. Adjust the position of the worm to a 20-40° angle relative to the needle by turning the gliding table (Figure 1A). The steep angle is necessary to prevent injuries in the cuticle.
    4. Switch to the 40x objective and focus on the cytoplasmic core of the distal germline of the animal. Then bring the tip of the needle close and in focus by using the micromanipulator in ‘fine’ mode.
    5. Move the animal by gently tapping the gliding table towards the tip of the needle. We move the animal into the needle, not the other way around, as this is easier and more precisely to handle. The needle tip has to be placed inside the cytoplasmic core of the distal gonad (Figures 1A and 1B).
    6. Press the ‘Inject’ button on the Microinjector unit to release a specified volume of DNA solution into the gonad. The liquid will slightly blow up the gonad (Video 1).

      Video 1. The injection procedure. The video shows (1) The localization of the needle and test release of injection mix, (2) Localization of the target animal and placing it into position for injection by rotating the gliding table, (3) Adjusting the needle position to target the gonad, (4) Switching to the 40x objective and centralize the position of the needle, (5) Focusing on the syncytium of the distal gonad, (6) Moving the animal into the needle and injecting the injection mix, as well as three more examples for the procedure.

    7. Gently move the gliding table in the opposite direction to remove the needle from the animal.
    8. Use the micromanipulator to move the needle up along the z-axis but leave it in the same x-y position for the next rounds of injection. Remove the agarose pad from the gliding table.
    9. Under the stereoscope, pipette a drop of 10 µl M9 buffer (see Recipes) on the injected animal to remove it from the agarose pad. Let the animal recover for 2 min in the M9 buffer before transferring it with a hairpin pick to an OP50 seeded NGM agar plate.
    10. Repeat the injection procedure with a new animal (go to step B5). Inject 20-25 animals in total. With increasing experience and injection speed, several animals can be immobilized on the agarose pad and injected sequentially. In that case, align the animals close to each other under the stereoscope. Upon successfully injecting the first animal move the injection needle out of focus up along the z-axis, move the gliding table to focus on the next animal (switching to the 10x objective might be required) and repeat the injection procedure.

  4. Selecting transgenic animals and maintaining stabile transgenic lines
    1. After the injection procedure grow the animals for 3-5 days at 20 °C. During that time, monitor the F1 progeny for the distinct phenotypic features caused by the expression of the co-transformation marker (see Table 1). This might require observation under a fluorescent stereomicroscope to detect expression of fluorescent proteins. The injection efficiency can broadly vary between 1-10% and more.
    2. Single out transgenic F1 progeny as soon as they reach the L4 or adult stage by picking them each to a new OP50 seeded NGM plates with a wormpick. Keep maintaining them at 20 °C and monitor the F2 generation for the transgenic phenotype. Approx. 5-10% of the transgenic F1 animals will stably inherit the extrachromosomal array in a non-Mendelian fashion. Hence, it is recommended to monitor at least 30-50 F1 transgenics for stabile inheritance.
    3. Stably inheriting transgenic lines have to be maintained by picking 10-30 transgenic animals to fresh OP50 seeded NGM plates every 3-5 days. The transmission efficiency across generations can vary as much as 5-80%. Maintain at least three independent stable lines for phenotypic analysis when over-expressing or ectopically expressing genes or dsRNA constructs. For CRISPR/Cas9 side-directed mutagenesis other maintenance and selection criteria will apply, which are summarized elsewhere (Dickinson and Goldstein, 2016).

Notes

  1. DNA samples: C. elegans can be transformed with any type of DNA including plasmid, fosmid or cosmid DNA, phage DNA, genomic DNA or linear DNA obtained from a PCR reaction. Generally, any commercial purification protocol can be used to clean up the DNA. We do not recommend protocols such as boiling lysis or phenol-chloroform extraction as the resulting DNA solution might contain chemicals that are toxic for the animals. A critical factor for successful transformation is the concentration of the DNA: We suggest to use 50-100 ng/µl per injected construct, which is sufficient to form an extrachromosomal array. In some cases, an extrachromosomal array can lead to lethality, which is often based on the dose-dependency of expression. This issue can often be overcome by lowering the concentration of the DNA construct, which leads to the formation of a smaller extrachromosomal array containing less gene copies (Mello et al., 1991). Generally, we inject DNA dissolved in distilled H2O or 1x TE buffer. A special microinjection buffer has been suggested to be advantageous in some cases, e.g., for direct injection into maturing oocyte nuclei (Fire, 1986).
  2. Microinjection capillaries: The injection needles can also be prepared with a needle puller (e.g., P-97 , Sutter Instrument, Novato, CA, USA). The main advantage is that the diameter and length of the tip can be adjusted to the desired dimensions. However, the tip of the needle has to be opened by carefully breaking it on the surface of the agarose pad or by dipping it into hydrofluoric acid.
  3. Extrachromosomal arrays obtained from microinjection can be integrated in the genome by causing chromosomal breaks through mutagenic chemicals (Ethylmethane Sulphonate, EMS) or ionizing radiation (Rieckher et al., 2009).
  4. The microinjection protocol can easily be adapted to inject a number of other chemicals, e.g., proteins, drugs or small RNA molecules in the gonad or other distinct tissues, such as the intestine.

Recipes

  1. OP50 seeded NGM agar plates
    1. For 1 L NGM agar, combine 3 g NaCl, 2.5 g Bacto peptone, 0.2 g streptomycin and 17 g agar. Autoclave for 30 min including stir bar and cool to 55 °C while stirring
    2. Next to a Bunsen burner, add 1 ml 1 M CaCl2 (stock: 14.7 g CaCl2 in 100 ml distilled H2O, autoclaved), 1 ml 1 M MgSO4 (stock: 12.03 g CaCl2 in 100 ml distilled H2O, autoclaved), 1 ml cholesterol (5 mg/ml in 100% ethanol), 1 ml nystatin (10 mg/ml in 70% ethanol) and 1 ml KPO4 (stock: 102.2 g KH2PO4 and 57.06 g K2HPO4 in 1 L distilled H2O, autoclaved)
    3. Use 60 mm Petri dishes and pour 11.5 ml of NGM agar per plate (adjust volume to size of Petri dishes). Let cool and harden for some hours
    4. Next to the Bunsen burner, streak a drop (~200 µl) of an Escherichia coli OP50 liquid culture in the center of each plate and grow at 37 °C for 8 h or at room temperature overnight
  2. 2% agarose pads for worm immobilization (prepare several in parallel)
    1. Weigh 2.0 g agarose in a cylindrical glass beaker
    2. Add 100 ml distilled H2O
    3. Heat in a microwave until close to boiling. Take out, stir with a pipette tip and boil again. Repeat until the agarose is dissolved
    4. On a clean bench, fix a microscope cover glass with two stripes of tape
    5. Put a drop (ca. 50 µl) of fresh 2% agarose solution in the middle of the cover glass
    6. Take a microscope slide and place it on top of the agarose drop. Gently press down to flatten the drop. The tape serves as spacer to give the agarose pad a specific thickness (Figure 4)
    7. Let the agarose harden for 30 sec. And remove the microscope slide
    8. Set a heat plate to 100 °C and put the agarose pad on top for approx. 1 min to dry the agarose. The slides can be prepared in advance and stored indefinitely


      Figure 4. Preparing of 2% agarose pad for microinjection. A. Use two stripes of tape to fix a coverglass to a clean work bench; B. Place a drop of about 50 µl freshly prepared 2% agarose in distilled H2O in the middle of the coverglass; C. Put a microscope slide on top of the agarose drop and gently press down to flatten it. Let the agarose solidify and remove the microscope slide.

  3. Hairpin pick
    Remove a hair from your eyebrows and glue it to the tip of a toothpick or a glass Pasteur pipette (Figure 5). Best is to prepare many of them with hair of different thickness and test the handling convenience. While using, sterilize the hairpin by dipping it into a 70% ethanol solution


    Figure 5. Preparing a hairpin pick for gentle manipulation of C. elegans. Remove a hair from your eyebrow and glue it to the tip of a glass Pasteur pipette or a toothpick.

  4. M9 buffer
    22 mM KH2PO4
    22 mM Na2HPO4
    85 mM NaCl
    1 mM MgSO4
    Combine in distilled H2O
    Sterilize by autoclaving for 15 min at 121 °C

Acknowledgments

This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme. This work has been adapted from Rieckher M., N. Kourtis N., Pasparaki A. and Tavernarakis N. (2009). ‘Transgenesis in Caenorhabditis elegans.’ Methods Mol Biol 561: 21-39.

References

  1. Carter, P. W., Roos, J. M. and Kemphues, K. J. (1990). Molecular analysis of zyg-11, a maternal-effect gene required for early embryogenesis of Caenorhabditis elegans. Mol Gen Genet 221(1): 72-80.
  2. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263(5148): 802-805.
  3. Dickinson, D. J. and Goldstein, B. (2016). CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics 202(3): 885-901.
  4. Fire, A. (1986). Integrative transformation of Caenorhabditis elegans. EMBO J 5(10): 2673-2680.
  5. Frokjaer-Jensen, C., Davis, M. W., Hopkins, C. E., Newman, B. J., Thummel, J. M., Olesen, S. P., Grunnet, M. and Jorgensen, E. M. (2008). Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet 40(11): 1375-1383.
  6. Granato, M., Schnabel, H. and Schnabel, R. (1994). pha-1, a selectable marker for gene transfer in C. elegans. Nucleic Acids Res 22(9): 1762-1763.
  7. Mello, C. and Fire, A. (1995). DNA transformation. Methods Cell Biol 48: 451-482.
  8. Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J 10(12): 3959-3970.
  9. Praitis, V., Casey, E., Collar, D. and Austin, J. (2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157(3): 1217-1226.
  10. Rieckher, M., Kourtis, N., Pasparaki, A. and Tavernarakis, N. (2009). Transgenesis in Caenorhabditis elegans. Methods Mol Biol 561: 21-39.
  11. Stinchcomb, D. T., Shaw, J. E., Carr, S. H. and Hirsh, D. (1985). Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol Cell Biol 5(12): 3484-3496.
  12. Tabara, H., Motohashi, T. and Kohara, Y. (1996). A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans. Nucleic Acids Res 24(11): 2119-2124.
  13. Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. and Driscoll, M. (2000). Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet 24(2): 180-183.

简介

显微注射是线虫秀丽隐杆线虫遗传转化中最常用的工具,促进基因的转基因表达,通过聚集的定期散布的短回文重复序列(CRISPR)-Cas9系统的基因组编辑或转录 dsRNA用于RNA干扰(RNAi)。 外源DNA被递送到成年雌雄同株的种系中的发育中的卵母细胞中,然后在它们的后代中产生转基因动物。 在该方案中,我们描述了显微注射程序和随后的转基因后代选择。
【背景】在C.通过显微注射的DNA转化通常用于产生过表达或异位表达可以与标签(例如,绿色荧光蛋白[GFP])融合的基因的转基因动物,允许突变体的表型拯救和/或蛋白质的定位和功能的分析(Carter等人,1990; Chalfie等人,1994; Mello和Fire,1995)。聚集的定期散布的短回文重复(CRISPR)-Cas9系统的出现需要显微注射以通过引入点突变或插入/缺失突变来实现高度特异性的基因组编辑(概述于Dickinson和Goldstein,2016)。此外,该技术被应用于dsRNA的可诱导和/或组织特异性转录以便于遗传性RNA干扰(RNAi)(Tavernarakis等人,2000) 。将遗传物质注入到成年雌雄同体动物的远端性腺合胞体中,其进入发育中的卵母细胞(图1)。外源DNA被排列成由50到300个拷贝的大的染色体外阵列,其以非孟德尔方式继承到后代。下一代后代(F1)以不同的概率携带染色体外阵列(5%至80%[Stinchcomb等人,1985; Mello et al。,1991] )。通过使用与感兴趣的DNA一起注入的共转化标记物,在后代中鉴定转基因动物(表1)。最常用的是绿色荧光蛋白(GFP)或红色荧光(mCherry)或优势rol-6(su1006)等位基因的咽表达,其在转基因F1后代中诱导出明显的滚动表型( Mello et al。,1991; Tabara等人,1996; Frokjaer-Jensen等人,2008)。

表1.具有不同表型的常用共转录标记,用于在显微注射后选择转基因动物。线虫



图1. C的显微注射方案。线 A.成人方案C.显示主要器官,包括咽,肠和性腺的线虫。当微注射时C线虫需要将注射毛细管插入远端性腺的合胞体(胞质核心)。镶嵌表示注射的兴趣区域。图1A所示区域的DIC图像。生殖细胞的细胞核清晰可见,并围绕合胞体。尺寸棒对应于50μm。

关键字:秀丽隐杆线虫, 遗传转化, 显微注射, 染色体外阵列, 生殖细胞系, GFP, 转基因动物

材料和试剂

  1. 无菌移液器吸头
  2. 微型移液器吸头(Eppendorf,目录号:5242956003)
  3. 显微注射毛细管(Eppendorf Femtotips II,0.5μm内径和0.7μm外径)(Eppendorf,目录号:930000043)
  4. 显微镜载玻片,76 x 26 mm(Carl Roth,目录号:0656.1)
  5. 显微镜罩眼镜,24 x 60毫米(VWR,目录号:631-1575)
  6. 胶带(〜1 mm厚)
  7. Greiner培养皿(60 x 15 mm)(Sigma-Aldrich,目录号:P5237)
  8. 玻璃巴斯德移液器,一次性,150毫米(BRAND,目录号:747715)
  9. ℃。 elegans 动物(例如,可从 遗传学中心 [CGC],美国明尼苏达州明尼苏达大学)
  10. 大肠杆菌OP50菌株(从Caenorhabditis 遗传学中心获得)
  11. 注射感兴趣的DNA(质粒,fosmid,PCR片段或类似物)和含有共同转录标记的质粒(参见表1)
  12. 用于DNA纯化的Miniprep试剂盒(例如,QIAGEN-tip 20)(QIAGEN,目录号:10023)
  13. 卤代烃油700(Sigma-Aldrich,目录号:H8898-100ML)
  14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  15. 蛋白胨(BD,Bacto TM ,目录号:211677)
  16. 硫酸链霉素盐(Sigma-Aldrich,目录号:S6501)
  17. 琼脂(Sigma-Aldrich,目录号:05040)
  18. 100%乙醇
  19. 氯化钙脱水(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080)
  20. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  21. 胆固醇储备溶液(SERVA Electrophoresis,目录号:17101.01)
  22. 制霉菌素储备溶液(Sigma-Aldrich,目录号:N3503)
  23. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:P018.1)
  24. 磷酸氢二钠(Na 2 HPO 4)(Carl Roth,目录号:T876.1)
  25. 磷酸氢二钾(K 2 HPO 4)(Carl Roth,目录号:5066.1)
  26. 琼脂糖(Biozym Scientific,目录号:840004)
  27. OP50种子NGM琼脂平板(参见食谱)
  28. 2%琼脂糖垫(见食谱)
  29. 发夹选择(见食谱)
  30. M9缓冲(见配方)

设备

  1. 本生燃烧器
  2. 圆柱形玻璃烧杯
  3. 带铂丝和预扁平针尖的蠕虫(Genesee Scientific,目录号:59-AWP和59-30P6)
  4. 微波
  5. 加热板(例如,,IKA,型号:C-MAG HP 4,目录号:0003581600)
  6. Puller(可选;例如,Sutter Instrument,型号:P-97)
  7. 高压灭菌器
  8. 立体显微镜(例如,Leica Microsystems,型号:Leica M80),任选的荧光立体显微镜(例如,Leica Microsystems,型号:Leica M165FC)
  9. 显微镜显微镜(ZEISS,型号:Axio Observer.A1配备差分干涉对比度[DIC]棱镜,滑翔台,物镜10x / 0.3 M27,物镜40x / 0.75 M27,可选相机AxioCam ERc 5秒,并监视观察和演示目的)
  10. 微操纵器(例如,Eppendorf InjectMan 4)(Eppendorf,目录号:5192000019)
  11. 微量注射器单元(例如,Eppendorf FemtoJet 4i)(Eppendorf,目录号:5252000013)
  12. 微量离心机(例如,Eppendorf离心机5424)(Eppendorf,型号:5424,目录号:5404000014)
  13. 分光光度计(例如,Thermo Fisher Scientific,型号:NanoDrop TM,目录号:ND-8000-GL)

程序

  1. 准备DNA和注射单位
    1. 按照QIAGEN-tip 20微量制备试剂盒的说明进行DNA纯化(见注释)。
    2. 在最后一步,用蒸馏的H 2 O 2洗脱DNA,用分光光度计测定浓度。
    3. 将100ng体积的50ng /μl感兴趣的DNA和共同转录标记(见表1)的混合物制备成100μl体积。
    4. 将微量离心机中的溶液以最大速度离心15分钟,优选在4℃。此步骤是必要的,以沉淀DNA溶液中的浮游颗粒,以防止显微注射毛细血管堵塞
    5. 从DNA溶液顶部吸取5μl至新的反应管,并直接用于显微注射。任何剩下的都可以在-20°C冷冻以备以后使用。
    6. 在注射程序前几分钟,打开显微镜显微注射,微量注射器单元和显微操纵器(见图2)。按照微量注射器单元的说明对系统进行加压和调整。设置"注入"功能来释放大约的音量。 10皮升(pl)。


      图2.线虫显微注射的设置的基本组件。 :一种。概述了设置及其所有组件:(a)具有10x和40x物镜和DIC光学元件的光学显微镜,(b)压力器具的微型注射器单元,(c1)所有三维(x,y,z轴)的显微操纵器控制),(c2)显微操纵器控制的毛细管(针)支架,(d)滑动台,以将固定的动物灵活地移动到针中,(e)立体显微镜,用于将动物安装在琼脂糖垫上并注射后恢复( f)可选地连接到相机用于记录和演示目的的监视器,(g)玻璃毛细管(注射针)。 B.滑动台(d),针架(c2)和针(g)的详细视图。 C.注射针上的特写镜头(Femtotips II,Eppendorf),可以用微型加样机吸头提示填充。

    7. 使用微型移液器吸头将1μl的干净的DNA溶液移液到显微注射毛细管中(见注释)。这个体积足以注射超过一百只动物。
    8. 小心地取出毛细管的盖子,并将其拧入显微操纵器的针架上。毛细血管力将收集针头尖端的溶液
  2. 准备注射动物
    1. 在注射程序前一天,使用立体镜和蠕虫将40只L4级动物从混合群体转移到新鲜OP50种子的NGM琼脂平板(参见食谱)。
    2. 在20°C将动物生长至具有完全发育的种系的年轻妊娠成虫(图1A)
    3. 在注射程序前一小时将动物转移到15°C。这降低了动物的敏捷性,使其更容易处理注射。
    4. 在琼脂糖区旁边的2%琼脂糖垫上放一滴约50μl卤代烃油(见食谱)。在立体镜下,将10-20个蠕虫转移到油中(将蠕虫浸入油中使得更容易转移蠕虫)。让动物在油中移动5-10分钟以去除附着于其角质层上的细菌,因为细菌残留物可以快速堵塞显微注射毛细管。
    5. 在琼脂垫表面放置另外一小滴约20μl卤代烃油。
    6. 使用发夹(请参阅食谱)将一个动物从琼脂糖旁边的卤代烃油转移到琼脂糖垫表面的滴液中。
    7. 使用立体镜的最大放大倍数轻轻按动发丝到琼脂糖表面。动物将附着在干琼脂糖表面上(图3)
    8. 将琼脂糖垫放置在显微镜的滑动台上以进行显微注射,并迅速开始注射程序,因为动物将在约10-15分钟

      图3.固定在2%琼脂糖垫上并准备注射的动物。箭头表示在性腺中注射的首选位点。刻度棒为50μm。

  3. 注射程序
    1. 使用10x目标将固定化的动物定位在琼脂糖垫上的卤代烃油滴中
    2. 使用"粗略"模式的显微操纵器沿着与蜗杆紧邻的xy轴移动注射毛细管的尖端,然后使用显微操纵器z轴控制的"精细"模式大致将针尖插入焦点(视频1)。
    3. 通过转动滑翔台,将蜗杆的位置调整到相对于针的20-40度角(图1A)。陡峭的角度是防止角质层受伤的必要条件
    4. 切换到40倍的目标,并集中在动物的远端种系的细胞质核心。然后使用"微调"模式下的显微操纵器,使针尖靠近并聚焦。
    5. 通过轻轻地将滑动的桌子朝向针尖移动动物。我们将动物移动到针头,而不是相反的方式,因为这更容易,更准确地处理。针头必须放置在远端性腺的细胞质核心内(图1A和1B)。
    6. 按微量注射器单元上的"注射"按钮将特定体积的DNA溶液释放到性腺中。 液体会稍微炸毁性腺(视频1)。

      Video 1. The injection procedure. The video shows (1) The localization of the needle and test release of injection mix, (2) Localization of the target animal and placing it into position for injection by rotating the gliding table, (3) Adjusting the needle position to target the gonad, (4) Switching to the 40x objective and centralize the position of the needle, (5) Focusing on the syncytium of the distal gonad, (6) Moving the animal into the needle and injecting the injection mix, as well as three more examples for the procedure.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    7. 轻轻地向相反方向移动滑翔台,以从动物身上取下针头。
    8. 使用显微操纵器将针沿z轴向上移动,但将其保持在相同的x-y位置,以进行下一轮注射。从滑翔表中取出琼脂糖垫。
    9. 在立体镜下,在注射的动物上移液一滴10μlM9缓冲液(参见食谱),将其从琼脂糖垫中取出。让动物在M9缓冲液中恢复2分钟,然后将其转发到OP50种子的NGM琼脂平板上。
    10. 用新动物重复注射程序(转到步骤B5)。总共注射20-25只动物。随着经验和注射速度的增加,几只动物可以固定在琼脂糖垫上并依次注射。在这种情况下,将动物在立体镜下相互靠近。成功注射第一只动物后,注射针头沿着Z轴移动聚焦,移动滑翔台以聚焦下一只动物(可能需要切换到10x物镜),并重复注射程序。

  4. 选择转基因动物并维持稳定的转基因品系
    1. 注射程序在20°C下将动物生长3-5天。在此期间,监测F1后代由共同转录标记表达引起的不同表型特征(见表1)。这可能需要在荧光立体显微镜下观察以检测荧光蛋白的表达。注射效率可以在1-10%之间变化。
    2. 通过将它们分别接种到具有蠕虫的新的OP50种子NGM板上,一旦它们到达L4或成年阶段,单独的转基因F1后代。将其保持在20°C,并监测F2代的转基因表型。约。 5-10%的转基因F1动物将以非孟德尔方式稳定地继承染色体外阵列。因此,建议监测至少30-50个F1转基因作为稳定遗传。
    3. 必须通过每3-5天通过选择10-30个转基因动物来维持新鲜的OP50种子NGM板来保持稳定的遗传转基因品系。几代人的传播效率可以变化多达5-80%。当过表达或异位表达基因或dsRNA构建体时,保持至少三个独立的稳定线用于表型分析。对于CRISPR / Cas9侧向诱变,其他维护和选择标准将适用,其他地方总结(Dickinson和Goldstein,2016)。

笔记

  1. DNA样品:C。线虫可以用任何类型的DNA转化,包括质粒,质粒或粘粒DNA,噬菌体DNA,基因组DNA或从PCR反应获得的线性DNA。通常,可以使用任何商业纯化方案来清除DNA。我们不推荐使用诸如沸腾裂解或苯酚 - 氯仿提取的方案,因为所得DNA溶液可能含有对动物有毒的化学物质。成功转化的关键因素是DNA的浓度:我们建议每个注射的构建体使用50-100 ng /μl,这足以形成染色体外阵列。在一些情况下,染色体外阵列可导致致死性,其通常基于表达的剂量依赖性。通常可以通过降低DNA构建体的浓度来克服这个问题,这导致形成含有较少基因拷贝的较小的染色体外阵列(Mello et al。,1991)。通常,我们注入溶解在蒸馏H 2 O或1×TE缓冲液中的DNA。已经提出特殊的显微注射缓冲液在某些情况下(例如,直接注射成熟的卵母细胞核)是有利的(Fire,1986)。
  2. 显微注射毛细管:注射针也可以用拔针器(例如,P-97,Sutter Instrument,Novato,CA,USA)来制备。主要的优点是尖端的直径和长度可以调节到所需的尺寸。然而,针头的尖端必须通过在琼脂糖垫的表面上小心地打破或将其浸入氢氟酸来打开。
  3. 由显微注射获得的染色体外阵列可通过致突变化学品(Ethylmethane Sulphonate,EMS)或电离辐射(Rieckher等人,2009)引起染色体断裂而整合到基因组中。
  4. 显微注射方案可以容易地适应于在性腺或其它不同组织例如肠中注射许多其它化学物质,例如蛋白质,药物或小RNA分子。

食谱

  1. OP50种子NGM琼脂平板
    1. 对于1L NGM琼脂,结合3g NaCl,2.5g细菌蛋白胨,0.2g链霉素和17g琼脂。高压灭菌30分钟,包括搅拌棒,冷却至55℃,同时搅拌
    2. 在本生灯附近,在100ml蒸馏的H 2 O 2中加入1ml 1M CaCl 2(储备:14.7g CaCl 2),高压灭菌),1ml 1M MgSO 4(储存液:12.03g CaCl 2在100ml蒸馏的H 2 O 2中,高压灭菌),1ml胆固醇(在100%乙醇中为5mg / ml),1ml制霉菌素(10mg / ml在70%乙醇中)和1ml KPO 4(储备:102.2g KH 2 / 1L蒸馏的H 2 O 3中的PO 4和57.06g K 2 HPO 4,高压灭菌) >
    3. 使用60毫米培养皿,每平板倒入11.5毫升NGM琼脂(调节体积至培养皿的大小)。让凉爽和硬化几个小时
    4. 在本生灯旁边,将大肠杆菌OP50液体培养物滴入(约200μl)的每个板中心,并在37℃下生长8小时或室温过夜/>
  2. 用于蠕虫固定的2%琼脂糖垫(并行准备几个)
    1. 称量2.0 g琼脂糖在圆柱形玻璃烧杯中
    2. 加入100ml蒸馏的H 2 O - / -
    3. 在微波炉中加热,直至接近沸腾。取出,用移液器吸头搅拌,再次煮沸。重复一遍直到琼脂糖溶解
    4. 在一个干净的工作台上,用两条胶带固定显微镜盖玻璃
    5. 将新鲜的2%琼脂糖溶液放在盖玻片中间( ca。 50μl)
    6. 拿一个显微镜载玻片放在琼脂糖滴下面。轻轻按下来压平下落。胶带用作间隔物以使琼脂糖垫具有特定的厚度(图4)
    7. 让琼脂糖硬化30秒。并移除显微镜幻灯片
    8. 将热板设置为100°C,将琼脂糖垫放在顶部约1分钟干燥琼脂糖。幻灯片可以提前准备并无限期存储


      图4.准备用于显微注射的2%琼脂糖垫。 A.使用两条胶带将盖玻片固定在干净的工作台上; B.将一滴约50μl新鲜制备的2%琼脂糖滴入蒸馏的H 2 O 2覆盖玻璃的中间; C.将显微镜载玻片放在琼脂糖滴上方,轻轻按压使其变平。让琼脂糖固化并除去显微镜载玻片。

  3. 发夹选择
    从眉毛上取下头发,然后将其粘到牙签或玻璃巴斯德吸管的尖端(图5)。最好是准备许多不同厚度的头发,并测试处理方便。在使用时,通过将发夹浸入70%乙醇溶液中进行消毒

    图5.准备一个发夹,轻轻操纵C。电线杆。从眉毛上取下头发,将其粘贴到玻璃巴斯德吸管或牙签的尖端。

  4. M9缓冲区
    22mM KH 2 PO 4
    22mM Na 2 HPO 4
    85 mM NaCl
    1mM MgSO 4
    在蒸馏的H 2 O结合在一起 在121℃下通过高压灭菌15分钟灭菌

致谢

这项工作是由欧洲研究委员会(ERC),欧洲委员会第七届支持框架计划的资助提供的。这项工作已经由Rieckher M.,N.Kourtis N.,Pasparaki A.和Tavernarakis N.(2009)进行了改编。 'Transgenesis in the Caenorhabditis elegans 。Methods Mol Biol 561:21-39。

参考

  1. Carter,P.W.,Roos,J.M.and Kemphues,K.J。(1990)。 zyg-11的分子分析,即切尔假丝酵母早期胚胎发生所需的母体效应基因elegans 。 Mol Gen Genet 221(1):72-80。
  2. Chalfie,M.,Tu,Y.,Euskirchen,G.,Ward,W.W.and Prasher,D.C。(1994)。 绿色荧光蛋白作为基因表达的标记。 科学 263(5148):802-805。
  3. Dickinson,D.J。和Goldstein,B。(2016)。 基因组工程中针对秀丽隐杆线虫的基于CRISPR的方法。遗传学 202(3):885-901。
  4. Fire,A.(1986)。 Caenorhabditis elegans 的整合式转换 EMBO J 5(10):2673-2680。
  5. Frokjaer-Jensen,C.,Davis,M.W.,Hopkins,C.E.,Newman,B.J.,Thummel,J.M.,Olesen,S.P.,Grunnet,M.and Jorgensen,E.M。(2008)。 转基因在<秀秀秀秀秀中的单拷贝插入 Nat Genet 40(11):1375-1383。
  6. Granato,M.,Schnabel,H。和Schnabel,R。(1994)。 pha-1 ,用于中的基因转移的选择标记C。 elegans 。 Nucleic Acids Res 22(9):1762-1763。
  7. Mello,C.和Fire,A.(1995)。 DNA转化。方法细胞周期48:451- 482.
  8. Mello,C.C.,Kramer,J.M.,Stinchcomb,D.and Ambros,V.(1991)。 C.elegans中有效的基因转移:染色体外维持和整合转化序列。 EMBO J 10(12):3959-3970。
  9. Praitis,V.,Casey,E.,Collar,D。和Austin,J.(2001)。 在秀丽隐杆线虫中创建低拷贝整合转基因株系。 遗传学 157(3):1217-1226。
  10. Rieckher,M.,Kourtis,N.,Pasparaki,A.and Tavernarakis,N。(2009)。 秀丽隐杆线虫中的转基因 方法Mol Biol 561:21-39。
  11. Stinchcomb,D.T.Shaw,J.E.,Carr,S.H。和Hirsh,D。(1985)。 秀丽隐杆线虫的染色体外DNA转化 Mol Cell Biol 5(12):3484-3496。
  12. Tabara,H.,Motohashi,T。和Kohara,Y。(1996)。 在秀丽隐杆线虫整个胚胎上的原位杂交的多孔版本 em>。 Nucleic Acids Res 24(11):2119-2124。
  13. Tavernarakis,N.,Wang,S.L.,Dorovkov,M.,Ryazanov,A。和Driscoll,M。(2000)。 由转基因编码的双链RNA可遗传和诱导的遗传干扰。 Nat Genet 24(2):180-183。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rieckher, M. and Tavernarakis, N. (2017). Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection. Bio-protocol 7(19): e2565. DOI: 10.21769/BioProtoc.2565.
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