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Oct 2018

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pNP Transgenic RNAi System Manual in Drosophila
果蝇中pNP RNAi 转基因系统手册   

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Abstract

Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.

Keywords: Drosophila (果蝇), pNP vector (pNP载体), Transgenic RNAi (转基因RNAi), Gal4/UAS system (Gal4/UAS系统), Multiple genes knockdown (多基因敲除)

Background

Drosophila melanogaster is easy to culture, and has a short life cycle, clear genetic background, and abundant research tools. More importantly, most genes in Drosophila are highly homologous to human genes, which over the past century made Drosophila a powerful model to study the genes in vivo.

RNAi technology can specifically reduce gene expression, which is efficient and easy to operate, so it has been widely used in different model organisms to study gene functions (Dietzl et al., 2007; Ni et al., 2008, 2009 and 2011; Perrimon et al., 2010). In Drosophila, transgenic RNAi approach is a binary system, where UAS-hairpin is controlled by GAL4 to achieve tissue- or developmental stage-specific gene knockout (Brand and Perrimon, 1993). Without Gal4, the hairpin downstream of the UAS will not be transcribed, thus transgenic hairpin lines behave similar to wild type. That is why we can keep thousands of transgenic RNAi lines. However, current RNAi systems exhibit many limitations, such as off-target effects, leaky expression of the basal promoter, poor efficiency to highly expressed genes and the unsolved challenge of simultaneously targeting multiple genes.

We developed a new transgenic RNAi system based on pNP vector, which has an intergenic linker between miR-2a-1 and miR-2b-2 and a modified promoter (Qiao et al., 2018). The pNP transgenic RNAi system can efficiently target highly expressed genes and produce expected severe phenotypes, which overcomes the poor efficiency of current RNAi systems (Ma et al., 2006; Markstein et al., 2008). Furthermore, the pNP transgenic RNAi system can innovatively knock down multiple genes simultaneously in one step, for example simultaneously depleting all the core components of PRC1 in the eye using the pNP system led to smaller eyes (Qiao et al., 2018). The efficiency of multiple mRNA depletion was further confirmed by qRT-PCR, which showed reduced expression levels of these targeted genes. Moreover, the loss-of-function phenotypes produced by targeting multiple genes simultaneously are more robust than the effect of sequentially depleting the genes. The following protocol details the steps from vector construction to transgenic RNAi flies’ screening, which gives us the approach to study the autonomous or non-autonomous roles of genes, including protein complexes or redundant genes.

Materials and Reagents

  1. 1.5 ml MaxyClear Microtubes (Axygen, catalog number: MCT-150-C)
  2. 0.2 ml Polypropylene PCR Tube (Axygen, catalog number: PCR-0208-C)
  3. Pipette tips (Corning, Axygen®)
  4. 0.22 μm Millipore filters (Biosharp, BS-QT-011)
  5. Petri dishes
  6. Drosophila lines (Tinghua Fly Center, http://fly.redbux.cn/)
    1. y[1] sc[1] v[1] p{y[+t7.7]}=nos-phiC31\int.NLS}X; P{y[+t7.7]=CaryP}attP40 (TB00016)
    2. y[1] sc[1] v[1] p{y[+t7.7]}=nos-phiC31\int.NLS}X; P{y[+t7.7]=CaryP}attP2 (TB00018)
    3. y[1] sc[1] v[1]; wg[Gla-1] Bc[1]/CyO (TB00023)
    4. y[1] sc[1] v[1]; Dr[1] e[1]/TM3, Sb[1] (TB00139)
    5. y[1] sc[1] v[1] (TB00077)
  7. pNP vector (Ampicillin resistant, plasmid map is shown in Figure 1. This vector is not commercial, but can be provided for free. If you want, please email your need to nijq@mail.tsinghua.edu.cn)


    Figure 1. The map of pNP construct. Vermilion is a selectable marker. attB sequence is used for phiC31 targeted integration at genomic attP landing sites. MCS1 allows a single shRNA to be cloned in both orientations, while with the help of MCS2 and the linker it could simultaneously generate multiple shRNAs once.

  8. EcoRI-HF (NEB, catalog number: R3101L)
  9. NheI-HF (NEB, catalog number: R3131L)
  10. SpeI (NEB, catalog number: R3133L)
  11. XbaI (NEB, catalog number: R0145L)
  12. Alkaline Phosphatase, Calf Intestinal (CIP, NEB, catalog number: M0290S)
  13. AxyPrepTM DNA Gel Extraction Kit (Corning, Axygen, catalog number: AP-GX-250)
  14. T4 DNA ligase (NEB, catalog number: M0202L)
  15. Trans5α Chemically Competent Cell (Transgen Biotech, catalog number: CD201-01)
  16. Go Tag Green Master Mix, 2x (Promega, catalog number: 000179370)
  17. AxyPrepTM Plasmid Miniprep Kit (Corning, Axygen, catalog number: AP-MN-P-250)
  18. PurePlasmid Mini Kit (CWBiotech, catalog number: CW0500M)
  19. Primers sequences:
    U-F: 5’-GCTGAGAGCATCAGTTGTGA-3’; Ftz: 5’-TAATCGTGTGTGATGCCTACC-3’
  20. Buffer PB (QIAGEN, catalog number: 154051779) 
  21. LB Broth, Miller (Luria Bertani) (Becton Dickinson)
  22. Agar (Beyotime)
  23. Ampicillin (Solarbio, catalog number: A8180)
  24. Tris-HCl (AMRESCO, catalog number: 1185-53-1)
  25. EDTA (AMRESCO, catalog number: 60-00-4)
  26. NaCl (AMRESCO, catalog number: 7647-14-5)
  27. KCl (AMRESCO, catalog number: 7447-40-7)
  28. Ethidium bromide (Sigma, catalog number: E8751)
  29. 50x TAE (Double Helix, catalog number: P0309A)
  30. Sodium phosphate buffer stock solution (pH 6.8) (see Recipes)
  31. 10x Annealing Buffer (see Recipes)
  32. Injection Buffer (see Recipes)
  33. LB (Luria Bertani) medium (see Recipes)
  34. LB/ampicillin plates (see Recipes)
  35. 1x TAE (see Recipes)
  36. 1.5% agarose gel (see Recipes)

Equipment

  1. Pipette (Eppendorf)
  2. Microwave (SANYO, model: EM-2509EB1)
  3. Autoclave (SANYO, model: MLS-3780)
  4. PCR Thermal Cycler (Eppendorf, Mastercycler nexus GSX1)
  5. Refrigerated centrifuge (Eppendorf, model: 5417R)
  6. Stereo Microscope (Olympus Corporation)
  7. NanoDrop 2000 (Thermo Scientific)

Software

  1. DSIR website: http://biodev.cea.fr/DSIR/DSIR.html
  2. FlyBase website: http://flybase.org/
  3. EMBOSS Needle: https://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html
  4. Tsinghua Fly Center: http://fly.redbux.cn

Procedure

  1. Vector construction
    1. Single short hairpin construction
      1. Short hairpins design
        Design primers for short hairpins (21 nt) on http://biodev.cea.fr/DSIR/DSIR.html website: first, copy the sequence of exon within the gene, and then paste the sequence to predict the siRNA with this web tool. Based on the parameters we defined (Figure 2), the siRNA with the highest score will be selected. To avoid the off-target effect, use BLAST to compare the siRNA against the fly genome of FlyBase website (http://flybase.org/) to ensure the 21 nt sequence has more than 5 nt that mismatch to other genes. “As re-com” sequence is sense sequence of the short hairpin. “As” sequence is the antisense sequence of the short hairpin.
        Primer-F: ctagcagt “As re-com” tagttatattcaagcata “As” gcg
        Primer-R: aattcgc “As re-com” tatgcttgaatataacta “As” actg


        Figure 2. Parameters for short hairpins design

      2. Annealing
        Perform the annealing reaction as follows, and then put the Eppendorf tube into PCR equipment. Denature the primers at 95 °C for 5 min, and then turn off the power of PCR equipment to let it cool down slowly to room temperature.


      3. Digestion
        Use EcoRI/NheI restriction endonucleases for pNP vector (Figure 1) digestion, and then separate the fragments with 1.5% agarose gels. Cut and purify the larger DNA fragment around 8,000 bp with AxyPrepTM DNA Gel Extraction Kit. Perform the digestion reaction as follows:


      4. Ligation
        Ligate the linearized pNP vector with the annealing product using T4 DNA ligase. Perform the ligation reaction as follows:


        Mix and incubate at 25 °C for 20 min, and then put 2-5 µl ligation product into 10 µl Trans5α Chemically Competent Cell following the standard protocol, and spread on an LB-agar plate containing 100 µg/ml ampicillin. Incubate the plate overnight at 37 °C, upside down.

      5. Select correct clones
        1. Mark and pick clones to the PCR mix. Primers sequences are U-F: 5’-GCTGAGAGCATCAGTTGTGA-3’, Ftz: 5’-TAATCGTGTGTGATGCCTACC-3’.


        2. Set up and run the PCR program as follows: 

        3. Identify the correct clones based on the size of the PCR product. By gel electrophoresis, the correct clones give around 750 bp bands, and the wrong clones appear either 1,000 bp or no band.
        4. Amplify the correct clone: Pick up the correct clone into 5 ml of LB medium containing 100 µg/ml ampicillin and incubate overnight at 37 °C at 250 rpm.
        5. Extract plasmid using AxyPrepTM Plasmid Miniprep Kit, and further confirm the right clone by DNA sequencing with the primer (U-F: 5’-GCTGAGAGCATCAGTTGTGA-3’).
    2. Construct multiple shRNAs in one vector
      1. For the two or more short hairpins in one vector, we first clone all short hairpins into the pNP vector respectively as mentioned above.
      2. Linearize vector pNP-shRNA1 using SpeI at 37 °C for 90 min as follows:


        Before gel electrophoresis, use CIP to treat the cutting product to dephosphorylating at 37 °C for 10 min as follows:


        Purify the product using AxyPrepTM DNA Gel Extraction Kit.

      3. Digest another hairpin vector using SpeI and XbaI to release the fragment (300 bp) containing a different short hairpin as follows:


        Separate the digested products on an agarose gel and extract smaller DNA fragment around 300 bp from the agarose gel with AxyPrepTM DNA Gel Extraction Kit. Finally, using T4 DNA ligase ligate the linearized pNP-shRNA1 with the released fragment (shRNA2). 
      4. Following transformation, use primers (U-F: 5’-GCTGAGAGCATCAGTTGTGA-3’, Ftz: 5’-TAATCGTGTGTGATGCCTACC-3’) to identify the correct clones by PCR, which give around 1,000 bp. Then further confirm these correct plasmids by DNA sequencing.
      5. To clone the third shRNA, the two shRNA containing vector is digested with SpeI again and then ligated with another short hairpin fragment released from hairpin vector by SpeI and XbaI digestion.

  2. Purify and inject the correct pNP-shRNA vector
    1. Purification of the plasmid DNA
      Extract plasmid DNA (~10 mg) using PurePlasmid Mini Kit as per the manufacturer’s instructions. Elute the plasmid DNA with 70 μl of 1x Injection Buffer. The appropriate concentration of each sample is 100-200 mg/ml, which is determined by using NanoDrop 2000.
    2. Injection of the plasmid DNA to flies
      Inject the purified plasmid DNA to flies (Chromosome II TB00016; Chromosome III TB00018) following the standard protocol (Ni et al., 2011). For knocking down a gene on the second chromosome, choose TB00016 to microinject, while a gene on the third chromosome to choose TB00018. Keep the injected embryos at 25 °C and 60% humidity to adult (G0).
    3. Selection protocols after G0 eclosion (Figures 3 and 4 are overviews of screening procedure)
      1. Once the G0 flies begin to hatch into adults, cross them to the TB00077 (vermillion eye color).
        Note: For injections into y,v stocks, G0s should all have vermillion eyes.


      Figure 3. Overview of the selection procedures for generating Chromosome II transgenic flies

      1. When adult G1 flies begin to eclose, screen G1 transformants. Remember that the pNP vector carry a copy of vermillion (v+), so G1 transformants will have a dark red wild type (v+) eye color.
      2. Cross the appropriate balancer stocks to ♂ G1 transformants from ♂ G0 parents (For Chromosome II integration into an attP40 site, G1 flies were crossed with TB00023. For Chromosome III integration into an attP2 site, G1 flies were crossed with TB00139). When G2 flies begin to eclose, collect ♂ and virgin ♀ siblings that have v+ (wild type) eye color and CyO (for Chromosome II insertions) or TM3 (for Chromosome III insertions), and cross siblings to each other to screen for homozygous transgenic flies. Collect ♂ G3 and virgin ♀ G3 siblings, and cross siblings to each other to make sure that the homozygous transgenic flies were fertile.
      3. ♂ transformants from ♀ G0 parents need to backcross TB00077 again to remove phiC31 integrase. Collect wild type (v+) eye color ♂ G2 transformants to cross the appropriate balancer stocks. The following sequential steps are consistent with the Step B3c.


      Figure 4. Overview of the selection procedures for generating Chromosome III transgenic flies

Data analysis

EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html) is used for sequence alignment. Detailed information on the data of this manual is available from the corresponding author upon reasonable request.

Recipes

  1. 10x Annealing Buffer (store at RT)

  2. Injection Buffer
    Prepare 1 mM sodium phosphate Buffer (pH 6.8) and 50 mM KCl in sterile bi-distilled water, and then filter through 0.22 μm Millipore filters, store at -20 °C
  3. Sodium phosphate buffer (pH 6.8) (stock solution)
    51 ml 0.2 M NaH2PO4
    49 ml 0.2 M Na2HPO4
    Store at room temperature
    1. 0.2 M Na2HPO4
      Dissolve 71.6 g Na2HPO4•12H2O in 1 L of ddH2O, mix thoroughly
      Store at room temperature
    2. 0.2 M NaH2PO4
      Dissolve 31.2 g Na2HPO4•2H2O in 1 L of ddH2O, mix thoroughly
      Store at room temperature
  4. LB (Luria Bertani) medium
    Dissolve 25 g of the powder in 1 L of ddH2O, mix thoroughly
    Autoclave at 121 °C for 15 min
  5. LB/ampicillin plates
    Add 25 g of LB media and 7 g Agar in 1 L of ddH2O
    Autoclave at 121 °C for 15 min
    After sterilization, allow the medium to cool before adding 100 µg/ml of ampicillin, pour 15 ml of medium into each Petri dish
  6. 1x TAE
    20 ml 50x TAE
    980 ml ddH2O
    Store at room temperature
  7. 1.5% agarose gel
    1.5 g agarose
    100 ml 1x TAE
    Heat solution to dissolve agarose in a microwave
    Add ethidium bromide to a final concentration of 0.2 μg/ml

Acknowledgments

This work was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China (2016YFE0113700), and the National Natural Science Foundation of China (31571320, 91729301, 81630103), and the National Science Fund for Distinguished Young Scholars (31725023).

Competing interests

The authors declare no competing financial interests.

References

  1. Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2): 401-415.
  2. Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S., Couto, A., Marra, V., Keleman, K. and Dickson, B. J. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448(7150): 151-156.
  3. Ni, J. Q., Liu, L. P., Binari, R., Hardy, R., Shim, H. S., Cavallaro, A., Booker, M., Pfeiffer, B. D., Markstein, M., Wang, H., Villalta, C., Laverty, T. R., Perkins, L. A. and Perrimon, N. (2009). A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182(4): 1089-1100.
  4. Ni, J. Q., Markstein, M., Binari, R., Pfeiffer, B., Liu, L. P., Villalta, C., Booker, M., Perkins, L. and Perrimon, N. (2008). Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods 5(1): 49-51.
  5. Ni, J. Q., Zhou, R., Czech, B., Liu, L. P., Holderbaum, L., Yang-Zhou, D., Shim, H. S., Tao, R., Handler, D., Karpowicz, P., Binari, R., Booker, M., Brennecke, J., Perkins, L. A., Hannon, G. J. and Perrimon, N. (2011). A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8(5): 405-407.
  6. Perrimon, N., Ni, J. Q. and Perkins, L. (2010). In vivo RNAi: today and tomorrow. Cold Spring Harb Perspect Biol 2(8): a003640.
  7. Qiao, H. H., Wang, F., Xu, R. G., Sun, J., Zhu, R., Mao, D., Ren, X., Wang, X., Jia, Y., Peng, P., Shen, D., Liu, L. P., Chang, Z., Wang, G., Li, S., Ji, J. Y., Liu, Q. and Ni, J. Q. (2018). An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila. Nat Commun 9(1): 4160.
  8. Ma, Y., Creanga, A., Lum, L., Beachy, P. A. (2006). Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443(7109): 359-63.
  9. Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E., Perrimon, N. (2008). Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40(4): 476-483.

简介

我们对生物过程潜在机制的大部分知识依赖于遗传方法,从而减少了基因活动,并详细分析了扰动的表型后果。 对于功能基因组研究,具体,系统和具有成本效益的方式至关重要。 转基因RNAi系统是研究基因功能的首要选择,因为其在果蝇中具有简单实用的特性。 我们建立了一种新的系统,该系统在体细胞和生殖细胞中均有效,并且具有高效和特异性。 通过该系统,我们可以精确有效地调节高表达基因,同时一步敲除多个基因。 在本研究中,我们提供了pNP系统的详细协议,该系统取代了其他转基因系统,并期望它可以为使用该系统的研究人员提供一些帮助。
【背景】 Drosophila melanogaster 易于培养,生命周期短,遗传背景清晰,研究工具丰富。更重要的是,果蝇中的大多数基因与人类基因高度同源,在过去的一个世纪里,它们使得果蝇成为研究体内基因的强大模型 >。

RNAi技术可以特异性地降低基因表达,这是有效且易于操作的,因此它已被广泛用于不同的模式生物中以研究基因功能(Dietzl et al。,2007; Ni et al。,2008,2009和2011; Perrimon et al。,2010)。在 Drosophila 中,转基因RNAi方法是二元系统,其中UAS-发夹由GAL4控制以实现组织或发育阶段特异性基因敲除(Brand和Perrimon,1993)。如果没有Gal4,UAS下游的发夹将不会被转录,因此转基因发夹线的行为类似于野生型。这就是为什么我们可以保留数千个转基因RNAi系。然而,目前的RNAi系统表现出许多局限性,例如脱靶效应,基础启动子的渗漏表达,高表达基因的效率差以及同时靶向多个基因的未解决的挑战。

我们开发了一种新的基于pNP载体的转基因RNAi系统,其具有miR-2a-1和miR-2b-2之间的基因间连接子和修饰的启动子(Qiao et al。,2018)。 pNP转基因RNAi系统可有效靶向高表达基因并产生预期的严重表型,克服了当前RNAi系统的低效率(Ma et al。,2006; Markstein et al。,2008)。此外,pNP转基因RNAi系统可以在一个步骤中创造性地同时敲除多个基因,例如同时耗尽眼睛中PRC1的所有核心成分,使用pNP系统导致眼睛变小(Qiao et al。,2018年)。通过qRT-PCR进一步证实了多mRNA消耗的效率,其显示这些靶基因的表达水平降低。此外,通过同时靶向多个基因产生的功能丧失表型比依次耗尽基因的效果更稳健。以下协议详述了从载体构建到转基因RNAi蝇筛选的步骤,这为我们提供了研究基因的自主或非自主作用的方法,包括蛋白质复合物或冗余基因。

关键字:果蝇, pNP载体, 转基因RNAi, Gal4/UAS系统, 多基因敲除

材料和试剂

  1. 1.5 ml MaxyClear Microtubes(Axygen,目录号:MCT-150-C)
  2. 0.2 ml聚丙烯PCR管(Axygen,目录号:PCR-0208-C)
  3. 移液器吸头(Corning,Axygen ®)
  4. 0.22μmMillipore过滤器(Biosharp,BS-QT-011)
  5. 培养皿
  6. Drosophila 系列(Tinghua Fly Center, http://fly.redbux.cn/ )
    1. y [1] sc [1] v [1] p {y [+ t7.7]} = nos-phiC31 \ int.NLS} X; P {y [+ t7.7] = CaryP} attP40 (TB00016)
    2. y [1] sc [1] v [1] p {y [+ t7.7]} = nos-phiC31 \ int.NLS} X; P {y [+ t7.7] = CaryP} attP2 (TB00018)
    3. y [1] sc [1] v [1]; wg [Gla-1] Bc [1] / CyO (TB00023)
    4. y [1] sc [1] v [1];博士[1] e [1] / TM3,Sb [1] (TB00139)
    5. y [1] sc [1] v [1] (TB00077)
  7. pNP载体(氨苄青霉素抗性,质粒图谱如图1所示。此载体不是商业化的,但可以免费提供。如果需要,请发送电子邮件至 nijq@mail.tsinghua.edu.cn )


    图1. pNP构建图。 Vermilion是一个可选择的标记。 attB序列用于基因组attP着陆点的phiC31靶向整合。 MCS1允许在两个方向上克隆单个shRNA,而在MCS2和连接子的帮助下,它可以同时产生多个shRNA。

  8. EcoRI-HF(NEB,目录号:R3101L)
  9. NheI-HF(NEB,目录号:R3131L)
  10. SpeI(NEB,目录号:R3133L)
  11. XbaI(NEB,目录号:R0145L)
  12. 碱性磷酸酶,小牛肠道(CIP,NEB,目录号:M0290S)
  13. AxyPrep TM DNA凝胶提取试剂盒(Corning,Axygen,目录号:AP-GX-250)
  14. T4 DNA连接酶(NEB,目录号:M0202L)
  15. Trans5α化学感受态细胞(Transgen Biotech,目录号:CD201-01)
  16. Go Tag Green Master Mix,2x(Promega,目录号:000179370)
  17. AxyPrep TM Plasmid Miniprep Kit(Corning,Axygen,目录号:AP-MN-P-250)
  18. PurePlasmid Mini Kit(CWBiotech,目录号:CW0500M)
  19. 引物序列:
    U-F:5'-GCTGAGAGCATCAGTTGTGA-3'; Ftz:5'-TAATCGTGTGTGATGCCTACC-3'
  20. Buffer PB(QIAGEN,目录号:154051779) 
  21. LB Broth,Miller(Luria Bertani)(Becton Dickinson)
  22. 琼脂(Beyotime)
  23. 氨苄青霉素(Solarbio,目录号:A8180)
  24. Tris-HCl(AMRESCO,目录号:1185-53-1)
  25. EDTA(AMRESCO,目录号:60-00-4)
  26. NaCl(AMRESCO,目录号:7647-14-5)
  27. KCl(AMRESCO,目录号:7447-40-7)
  28. 溴化乙锭(Sigma,目录号:E8751)
  29. 50x TAE(双螺旋,目录号:P0309A)
  30. 磷酸钠缓冲储备液(pH 6.8)(见食谱)
  31. 10x退火缓冲液(见食谱)
  32. 注射缓冲液(见食谱)
  33. LB(Luria Bertani)中等(见食谱)
  34. LB /氨苄青霉素板(见食谱)
  35. 1x TAE(见食谱)
  36. 1.5%琼脂糖凝胶(见食谱)

设备

  1. 移液器(Eppendorf)
  2. 微波炉(三洋,型号:EM-2509EB1)
  3. 高压灭菌器(三洋,型号:MLS-3780)
  4. PCR热循环仪(Eppendorf,Mastercycler nexus GSX1)
  5. 冷藏离心机(Eppendorf,型号:5417R)
  6. 立体显微镜(奥林巴斯公司)
  7. NanoDrop 2000(Thermo Scientific)

软件

  1. DSIR网站: http://biodev.cea.fr/DSIR/DSIR.html
  2. FlyBase网站: http://flybase.org/
  3. EMBOSS针: https://www.ebi.ac.uk/Tools/ PSA / emboss_needle / nucleotide.html
  4. 清华飞行中心: http://fly.redbux.cn

程序

  1. 矢量建筑
    1. 单发短发构造
      1. 短发夹设计
        在 http://biodev.cea.fr/DSIR/DSIR上设计短发夹(21 nt)的引物.html 网站:首先,复制基因内的外显子序列,然后粘贴序列以使用此网络工具预测siRNA。基于我们定义的参数(图2),将选择具有最高分数的siRNA。为了避免脱靶效应,使用BLAST比较siRNA与FlyBase网站的苍蝇基因组( http://flybase.org/ )确保21 nt序列具有超过5 nt与其他基因不匹配的序列。 “作为re-com”序列是短发夹的有义序列。 “As”序列是短发夹的反义序列。
        Primer-F:ctagcagt“as re-com”tagttatattcaagcata“As”gcg
        Primer-R:aattcgc“as re-com”tatgcttgaatataacta“As”actg


        图2.短发夹设计的参数

      2. 退火
        如下进行退火反应,然后将Eppendorf管放入PCR设备中。将引物在95°C下变性5分钟,然后关闭PCR设备的电源,让其缓慢冷却至室温。


      3. 消化
        使用EcoRI / NheI限制性内切核酸酶进行pNP载体(图1)消化,然后用1.5%琼脂糖凝胶分离片段。用AxyPrep TM DNA凝胶提取试剂盒切割并纯化大约8,000bp的较大DNA片段。如下进行消化反应:


      4. 结扎
        使用T4 DNA连接酶用退火产物连接线性化的pNP载体。如下进行连接反应:


        混合并在25℃下孵育20分钟,然后按照标准方案将2-5μl连接产物加入10μlTrans5α化学感受态细胞中,并涂布在含有100μg/ ml氨苄青霉素的LB琼脂平板上。将板在37°C孵育过夜,颠倒。

      5. 选择正确的克隆
        1. 标记并挑选PCR混合物的克隆。引物序列是U-F:5'-GCTGAGAGCATCAGTTGTGA-3',Ftz:5'-TAATCGTGTGTGATGCCTACC-3'。


        2. 按如下方式设置和运行PCR程序: 

        3. 根据PCR产物的大小确定正确的克隆。通过凝胶电泳,正确的克隆产生约750bp的条带,错误的克隆出现1,000bp或无条带。
        4. 扩增正确的克隆:将正确的克隆装入5ml含有100μg/ ml氨苄青霉素的LB培养基中,并在37℃,250rpm下孵育过夜。
        5. 使用AxyPrep TM 质粒Miniprep试剂盒提取质粒,并通过用引物(U-F:5'-GCTGAGAGCATCAGTTGTGA-3')进行DNA测序进一步确认正确的克隆。
    2. 在一个载体中构建多个shRNA
      1. 对于一个载体中的两个或更多个短发夹,我们首先将所有短发夹分别克隆到pNP载体中,如上所述。
      2. 使用SpeI在37℃下将载体pNP-shRNA1线性化90分钟,如下:


        在凝胶电泳前,使用CIP处理切割产品,在37°C下进行10分钟的去磷酸化,如下所示:


        使用AxyPrep TM DNA凝胶提取试剂盒纯化产物。

      3. 使用SpeI和XbaI消化另一个发夹载体,释放含有不同短发夹的片段(300 bp),如下所示:


        将消化的产物分离在琼脂糖凝胶上,用AxyPrep TM DNA凝胶提取试剂盒从琼脂糖凝胶中提取约300bp的较小DNA片段。最后,使用T4 DNA连接酶将线性化的pNP-shRNA1与释放的片段(shRNA2)连接。 
      4. 转化后,使用引物(U-F:5'-GCTGAGAGCATCAGTTGTGA-3',Ftz:5'-TAATCGTGTGTGATGCCTACC-3')通过PCR鉴定正确的克隆,其产生约1,000bp。然后通过DNA测序进一步确认这些正确的质粒。
      5. 为了克隆第三shRNA,将含有两个shRNA的载体再次用SpeI消化,然后通过SpeI和XbaI消化与发夹载体释放的另一个短发夹片段连接。

  2. 纯化并注射正确的pNP-shRNA载体
    1. 纯化质粒DNA
      按照制造商的说明,使用PurePlasmid Mini Kit提取质粒DNA(~10 mg)。用70μl1x注射缓冲液洗脱质粒DNA。每种样品的适当浓度为100-200mg / ml,其通过使用NanoDrop 2000测定。
    2. 向苍蝇注射质粒DNA
      按照标准方案(Ni et al。,2011)将纯化的质粒DNA注射到果蝇(染色体II TB00016;染色体III TB00018)中。为了敲除第二条染色体上的基因,选择TB00016进行显微注射,而选择第三条染色体上的基因选择TB00018。将注射的胚胎保持在25°C和60%湿度至成人(G0)。
    3. G0羽化后的选择方案(图3和图4是筛选程序的概述)
      1. 一旦G0苍蝇开始孵化成成虫,将它们交叉到TB00077(朱红色的眼睛颜色)。
        注意:对于y,v股票的注入,G0应该都有朱红色的眼睛。


      图3.产生染色体II转基因果蝇的选择程序概述

      1. 当成年G1蝇开始闭合时,筛选G1转化体。请记住,pNP载体携带 vermillion ( v + )的副本,因此G1转化体将具有深红色野生型( v + )眼睛颜色。
      2. 将适当的平衡剂原种与来自♂G0亲本的♂G1转化体杂交(对于染色体II整合到attP40位点,G1蝇与TB00023杂交。对于染色体III整合到attP2位点,G1蝇与TB00139杂交)。当G2苍蝇开始闭合时,收集具有 v + (野生型)眼睛颜色和CyO(对于染色体II)的♂和处女♀兄弟姐妹插入)或TM3(用于染色体III插入),并且彼此交叉兄弟以筛选纯合的转基因果蝇。收集♂G3和处女♀G3兄弟姐妹,并互相交叉兄弟姐妹,以确保纯合的转基因果蝇是可育的。
      3. ♂来自♀G0父母的转化体需要再次回交TB00077以去除phiC31整合酶。收集野生型( v + )眼睛颜色♂G2转化体以穿过适当的平衡器种群。以下顺序步骤与步骤B3c一致。


      图4.产生染色体III转基因果蝇的选择程序概述

数据分析

EMBOSS针( https://www.ebi.ac.uk/Tools/ psa / emboss_needle / nucleotide.html )用于序列比对。有关本手册数据的详细信息,可在合理要求下从相应作者处获得。

食谱

  1. 10x退火缓冲液(在RT存储)

  2. 注射缓冲液
    在无菌双蒸水中制备1 mM磷酸钠缓冲液(pH 6.8)和50 mM KCl,然后通过0.22μmMillipore过滤器过滤,储存于-20°C
  3. 磷酸钠缓冲液(pH 6.8)(原液)
    51ml 0.2M NaH 2 PO 4
    49ml 0.2M Na 2 HPO 4
    在室温下储存
    1. 0.2 M Na 2 HPO 4
      在1L ddH 2 O中溶解71.6g Na 2 HPO 4 •12H 2 O,充分混合< br /> 在室温下储存
    2. 0.2M NaH 2 PO 4
      在1L ddH 2 O中溶解31.2g Na 2 HPO 4 •2H 2 O,充分混合< br /> 在室温下储存
  4. LB(Luria Bertani)中等
    将25g粉末溶于1L ddH 2 O中,充分混合
    在121℃下高压灭菌15分钟
  5. LB /氨苄青霉素板
    在1升ddH 2 O中加入25克LB培养基和7克琼脂。 在121°C高压灭菌15分钟
    灭菌后,让培养基冷却,然后加入100μg/ ml的氨苄青霉素,将15ml培养基倒入每个培养皿中
  6. 1x TAE
    20毫升50倍TAE
    980 ml ddH 2 O
    在室温下储存
  7. 1.5%琼脂糖凝胶
    1.5克琼脂糖
    100毫升1x TAE
    加热溶液以在微波炉中溶解琼脂糖
    加入溴化乙锭至终浓度为0.2μg/ ml

致谢

这项工作得到了中华人民共和国科学技术部国家重点技术研究发展计划(2016YFE0113700)和国家自然科学基金(31571320,91729301,81630103)和国家科学部的支持。杰出青年学者基金(31725023)。

利益争夺

作者声明没有竞争性的经济利益。

参考

  1. Brand,A。H.和Perrimon,N。(1993)。 有针对性的基因表达作为改变细胞命运和产生显性表型的手段。 发展 118(2):401-415。
  2. Dietzl,G.,Chen,D.,Schnorrer,F.,Su,KC,Barinova,Y.,Fellner,M.,Gasser,B.,Kinsey,K.,Oppel,S.,Scheiblauer,S.,Couto ,A.,Marra,V.,Keleman,K。和Dickson,BJ(2007)。 用于条件基因失活的全基因组转基因RNAi文库 Drosophila 。 自然 448(7150):151-156。
  3. Ni,JQ,Liu,LP,Binari,R.,Hardy,R.,Shim,HS,Cavallaro,A.,Booker,M.,Pfeiffer,BD,Markstein,M.,Wang,H.,Villalta,C。 ,Laverty,TR,Perkins,LA和Perrimon,N。(2009)。 用于神经遗传学的转基因RNAi系的 Drosophila 资源。 Genetics 182(4):1089-1100。
  4. Ni,J.Q.,Markstein,M.,Binari,R.,Pfeiffer,B.,Liu,L.P.,Villalta,C.,Booker,M.,Perkins,L。和Perrimon,N。(2008)。 Drosophila melanogaster 中靶向转基因RNA干扰的载体和参数。 Nat Methods 5(1):49-51。
  5. Ni,JQ,Zhou,R.,Czech,B.,Liu,LP,Holderbaum,L.,Yang-Zhou,D.,Shim,HS,Tao,R.,Handler,D.,Karpowicz,P.,Binari ,R.,Booker,M.,Brennecke,J.,Perkins,LA,Hannon,GJ和Perrimon,N。(2011)。 在果蝇中转基因RNAi的基因组规模shRNA资源。 Nat方法 8(5):405-407。
  6. Perrimon,N.,Ni,J.Q。和Perkins,L。(2010)。 体内 RNAi:今天和明天。 Cold Spring Harb Perspect Biol 2(8):a003640。
  7. Qiao,HH,Wang,F.,Xu,RG,Sun,J.,Zhu,R.,Mao,D.,Ren,X.,Wang,X.,Jia,Y.,Peng,P.,Shen, D.,Liu,LP,Chang,Z.,Wang,G.,Li,S.,Ji,JY,Liu,Q。和Ni,JQ(2018)。 在果蝇中具有低毒性的高效多目标转基因RNAi技术。 Nat Commun 9(1):4160。
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  9. Markstein,M.,Pitsouli,C.,Villalta,C.,Celniker,S.E.,Perrimon,N。(2008)。 利用位置效应和吉普赛逆转录病毒绝缘体来设计精确表达的转基因。 Nat Genet 40(4):476-483。
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引用:Wang, F., Qiao, H., Xu, R., Sun, J., Zhu, R., Mao, D. and Ni, J. (2019). pNP Transgenic RNAi System Manual in Drosophila. Bio-protocol 9(3): e3158. DOI: 10.21769/BioProtoc.3158.
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