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Feb 2020

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Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila
RNAi介导的基因敲除后果蝇的分子和表型特征   

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Abstract

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency.


RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

Keywords: dElys (dElys), Nucleoporins (核孔蛋白), RNAi (RNA干扰), UAS-Gal4 (UAS-Gal4), Drosophila melanogaster (果蝇), Semiquantitative PCR (半定量PCR), Nuclear pores (核孔)

Background

Drosophila melanogaster (fruit fly) is a versatile model organism frequently used in research laboratories. Fruit flies are easy to handle, propagate, and maintain. Moreover, the elaborate yet short life span with high fecundity is an added advantage of Drosophila. The facile nature of Drosophila genetics tools helps develop a comprehensive understanding of a gene function. Since 60% of the Drosophila genes are homologous to human genes, and other advantages mentioned earlier, Drosophila is an obvious model organism of choice to study in vivo gene functions.


The UAS-GAL4 system was used for in vivo activation of transcription of genes. Moreover, it was suggested that antisense RNAs could be expressed using the UAS-GAL4 system to achieve significant inhibition of gene expression (Brand and Perrimon, 1993). For using the UAS-GAL4 system in Drosophila, transgenic fly lines carrying the upstream activating sequence (UAS) capable of regulating RNAi expression are generated. Genome-scale shRNA-dependent RNAi resource was generated for stage-specific effective knockdown of Drosophila genes (Ni et al., 2011). RNAi lines are publicly available from Vienna Drosophila Resource Center (VDRC, maintaining GD and KK RNAi lines) and Drosophila RNAi Screening Center (TRiP and shRNA lines were generated at DRSC and are currently maintained and available from Bloomington Drosophila Stock Center, BDSC). The detailed methodology for the generation of different RNAi lines can be found at the VDRC stock center (https://stockcenter.vdrc.at/control/main) and DRSC (https://fgr.hms.harvard.edu/) websites.


When the RNAi fly line is crossed with the GAL4 driver fly line, the progenies can have both components of the UAS-GAL4 system expressed. The upstream promoter guides GAL4 expression, and wherever the GAL4 is expressed, it binds to the UAS sequence with high affinity, allowing expression of the RNAi component (Figure 1). In the absence of GAL4 expression, the transgenic RNAi flies behave like wild-type due to lack of dsRNA expression. This method allows the knockdown of the desired gene in a spatiotemporal manner. Classically, X-ray mutagenesis, and P-element mobilization have been used to achieve the loss of gene expression. Further, the EMS mutagenesis screens have been utilized to induce point mutation, occasionally leading to truncations affecting gene expression. The RNAi mediated knockdown is rather facile and less labor-intensive.



Figure 1. Schematic diagram detailing the functioning of the UAS-GAL4 system in Drosophila Due to varying GAL4 expression degrees in different tissues, a gradient in the knockdown can be seen in different tissues. Besides, temperature-dependent regulation on the GAL4 function provides an advantage of regulating knockdown levels. Together, these handles on gene expression regulation using the UAS-GAL4 system provide more information regarding the functions of our gene of interest. Importantly, this method provides a controlled spatiotemporal regulation on RNAi mediated knockdown and can help study functions of developmentally essential genes, genes with housekeeping functions, and mutations in genes associated with harmful consequences. This method also helps in the functional characterization of a new gene like dElys (Mehta et al., 2020).


A typical qRT-PCR or semiquantitative PCR validates the reduction in gene expression levels upon RNAi mediated knockdown. The knockdown efficiencies can be further assessed by detecting the desired gene product levels by immunostaining and Western blotting methods. We have utilized the RNAi mediated knockdown in salivary glands and assessed its effectiveness using immunostaining or assessing mRNA levels by semiquantitative PCR. We successfully depleted nucleoporins in eyes and wings using the RNAi-mediated knockdown method and reported the importance of nucleoporins in tissue and organism development.


Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Genaxy, catalog number: GEN-MT-150-C-S)

  2. Barrier tips

    1. 0.5-10 µl tips (Axygen Scientific, catalog number: TF-300-L-R-S)

    2. 1-20 µl tips (Axygen Scientific, catalog number: TF-20-L-R-S)

    3. 1-200 µl tips (Axygen Scientific, catalog number: TF-200-L-R-S)

    4. 100-1,000 µl tips (Axygen Scientific, catalog number: TF-1000-L-R-S)

  3. Glass vials and bottles

  4. SYLGARD 184 Silicone elastomer (DOW, catalog number: 1673921)

  5. Micropipette tips (2, 20, 200, 1,000 µl)

    1. 0.5-10 µl tips (Axygen Scientific, catalog number: T-300-L-R-S)

    2. 1-200 µl tips (Axygen Scientific, catalog number: TR-222-C-L-R-S)

    3. 100-1,000 µl tips (Axygen Scientific, catalog number: T-1000-C-L-R-S)

  6. 3 cm dish (Eppendorf, catalog number: 0030700112)

  7. Food bottles

  8. CO2 pads

  9. Cotton plugs

  10. Corn flour

  11. Table sugar

  12. Yeast extract (HiMedia, catalog number: RM027)

  13. Agar (Merck, catalog number: A5306)

  14. Dextrose (HiMedia, catalog number: GRM077)

  15. Methyl-4-Hydroxybenzoate (Sigma-Aldrich, catalog number: H5501)

  16. Ortho-Phosphoric Acid (Merck, catalog number: 100573)

  17. Propionic Acid (HiMedia, catalog number: GRM3658)

  18. Ethanol (Changshu Hongsheng Fine Chemical CHN01)

  19. Liquid Nitrogen

  20. RNA isolation kit (Favorgen, catalog number: FATRK001)

  21. RNaseZAP (Thermo Scientific, catalog number: AM9780)

  22. DNase (Thermo Scientific, catalog number: EN0523)

  23. Diethyl pyrocarbonate (HiMedia, catalog number: MB076)

  24. Ethanol (MP Biomedicals, catalog number: 180077)

  25. Formamide (Sigma-Aldrich, catalog number: F7503)

  26. 6× DNA loading dye purple (New England Biolabs, catalog number: B7024S)

  27. cDNA synthesis kit (iScript, Bio-Rad, catalog number: 1708891)

  28. Agarose (Invitrogen, catalog number: 16500500)

  29. Ethidium bromide (EtBr) (MP Biomedicals, catalog number: 193993)

  30. Custom Primers (Integrated DNA Technology)

  31. G9-Taq DNA polymerase (GCC Biotech, catalog number: G7115A)

  32. dNTPs solution mix (New England Biolabs, catalog number: N0447L)

  33. Sodium Chloride (Sigma-Aldrich, catalog number: S3014)

  34. Potassium Chloride (Sigma-Aldrich, catalog number: P5405)

  35. di-Sodium hydrogen phosphate heptahydrate (Merck, catalog number: 1065751000)

  36. Potassium dihydrogen phosphate (Sigma-Aldrich, catalog number: P9791)

  37. Tris-base (Sigma-Aldrich, catalog number: T6066)

  38. Glacial acetic acid (Merck EMPLURA, catalog number: 1.93402.2521)

  39. Carbon conductive tape (TED PELLLA Inc. catalog number: 16073-1)

  40. Aluminum stub (TED PELLLA Inc. catalog number: 16111-9)

  41. Glutaraldehyde (Sigma-Aldrich, catalog number: G5882)

  42. Gold target (Quorum Technology inc, Catalog number: SC502-314A)

  43. EDTA (Merck EMPARTA, catalog number: 1.93312.1021)

  44. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127)

  45. Triton X-100 (Sigma-Aldrich, catalog number: X100)

  46. Glass Slide (Borosil, catalog number: 9100P02)

  47. Coverslips (Borosil, catalog number: 9115S01)

  48. Bovine serum albumin (Merck, catalog number: 621650500501730)

  49. Fluoroshield with DAPI (Sigma-Aldrich, catalog number: F6057)

  50. mAb414 (Biolegend 902901) - mAb414 recognizes FG-repeat rich four distinct nucleoporins of nuclear pores

  51. Anti-dElys (Mehta et al, 2020)

  52. Alexa Fluor-488 (Invitrogen, catalog number: A-11029)

  53. Alexa Fluor-568 (Invitrogen, catalog number: A-11036)

  54. Coverslip sealant (Transparent nail polish)

  55. Diethyl ether (Merck, catalog number: 107026)

  56. Phosphate Buffer Saline (1×) (see Recipes)

  57. PBS-T (see Recipes)

  58. Fly food ingredients (see Recipes)

  59. RNA loading dye (see Recipes)

  60. DEPC treated water (see Recipes)

  61. TAE Buffer (50×) (see Recipes)

  62. 4% Paraformaldehyde (see Recipes)


Fly stocks

Control Wild type (w1118)

Driver lines (in house generated combinations)

  1. +/+; Actin5C-GAL4/CyO-GFP; UAS-Dicer/UAS-Dicer

  2. +/+; wingless-GAL4/wingless-GAL4; UAS-Dicer/UAS-Dicer

  3. +/+; eyeless-GAL4/eyeless-GAL4; UAS-Dicer/UAS-Dicer

RNAi lines
  1. +/+; v103547/v103547; UAS-Dicer/UAS-Dicer (dElys RNAi KK line combined with UAS-Dicer, in house generation)

  2. dElys RNAi KK line (VDRC, VDRC ID: 103547)

  3. Nup160 RNAi GD line (VDRC, VDRC ID: 21937)

  4. Sec13 RNAi KK line (VDRC, VDRC ID: 110428)

  5. Nup107 RNAi GD line (VDRC, VDRC ID: 22407)

Equipment

  1. Micropipette (Nichiriyo, Gilson, Corning)

  2. BOD Incubators

  3. Millipore water purification unit

  4. Genova Nanodrop (Bibby Scientific, model: 737501)

  5. Forceps Dumont 5 (Fine Science Tools, catalog number: 11295-10)

  6. Forceps Dumont 55 (Fine Science Tools, catalog number: 11295-51)

  7. Scissors (Fine Science Tools, catalog number: 91500-09)

  8. Leica DM2500

  9. Fluorescent stereomicroscope (Leica, model: M205 FA)

  10. Confocal Laser Scanning Microscope (Zeiss, model: LSM780)

  11. Stereomicroscope (Leica, model: S6E)

  12. Scanning Electron microscope (Zeiss, model: Gemini II Ultra plus) <

  13. Tabletop centrifuge (Eppendorf, model: 5424, Thermo Scientific MicroCL 21R)

  14. PCR machine (Applied Biosystems, model: 2720 Thermal cycler)

  15. Water bath (Grant-Bio, model: PSU-10i)

  16. Gel running apparatus (CBS Scientific, model: GCMGU-202T)

  17. Power packs (CBS Scientific, model: EPS 300)

  18. UVP MultiDoc-It Digital Imaging System (UVP, catalog number: 97-0192-02)

Software

  1. ZEN 3.2 Blue edition (Zeiss)

  2. ImageJ/Fiji (Free, NIH)

Procedure

The UAS-GAL4 dependent and RNAi mediated knockdown of genes in Drosophila requires several steps. It includes steps like rearing the organism, setting up genetic crosses, harvesting larvae of desired genetic combination, RNA isolation and knockdown assessment, dissections, immunostaining, and phenotypic analysis. Mentioned below is the general scheme of tissue preparation and processing (Figure 2).



Figure 2. A flow chart with details of the protocol

  1. Genetic crosses for RNAi mediated knockdown

    1. Grow +/+; Actin5C-GAL4/CyO-GFP; UAS-Dicer/UAS-Dicer flies in large numbers in vials containing fly media.

    2. Collect virgin females having the GAL4 driver in a separate media vial.

    3. Grow RNAi and wild type (w1118) flies in separate media vials to get sufficient male flies.

    4. Place 6-7 virgin driver females and 2-3 UAS-RNAi males of the same age (3-4 days old) inside a fresh media vial to establish a mating cross.

    5. For the control cross, use w1118 males instead of UAS-RNAi males and set up the cross with virgin females as in Step A4.

    6. Incubate vials with flies for the genetic cross at room temperature for 24 h.

    7. According to the experimental design, shift them to 28 °C, 25 °C, or 18 °C.

      Note: Incubation at a higher temperature allows higher GAL4 activity inducing increased RNAi expression.

    8. Keep crosses in an incubator set at the desired temperature until the third instar larvae are visible.

    9. Remove adult flies and put 2-3 ml water or 2% sucrose solution to harvest larvae from the media's top layer.

      Note: Steps A8 to A13 are in Figure 3.

    10. Using a paintbrush, mix the upper layer of food and water and collect it in a clean plastic dish.

    11. Separate third instar larvae in a clean distilled water/phosphate buffer saline containing plate.

    12. Wash collected larvae 2-3 times with water to clean them.

    13. Segregate non-GFP larvae (+/+; Actin5C/RNAi; UAS-Dicer/+, indicating the desired genetic combination of Driver and RNAi) from GFP (+/+; RNAi/CyoGFP; UAS-Dicer/+) larvae for subsequent processing.

    14. For the control, use non-GFP larvae from the control cross as described in Step A5.

    15. Dissect the salivary gland or the head complex of Actin5C-GAL4 driven RNAi from the non-GFP third instar larvae in PBS.

      Note: Refer to Figure 3 below and Steps 1-5 mentioned in Part-C for details of salivary gland dissection.



      Figure 3. Steps in the isolation of Salivary glands from Drosophila third instar larva. Upper panels (a-f), Steps in larva separation and cleaning; (a) Larvae and Pupae in food vial, (b) Squirting water and suspending the top layer of food and larva with a brush, (c) Decanting suspension in a petri-plate, (d) Fly food and third instar larva suspension, (e) Cleaned larva collected in distilled water, (f) A third instar larva. Lower panels (g-l) Steps in larval dissection and salivary gland isolation (g) A third instar larva, (h) Holding a third instar larva with forceps, (i) Pulled out salivary glands and abdominal parts of the third instar larva, (j) Head complex having the brain, imaginal disc, and salivary gland (k) Salivary gland with associated fat bodies, (l) Dissected and cleaned up pair of salivary glands with an attached mouth hook.


  2. RNA isolation

    The most critical step of this method is RNA isolation. RNAi mediated knockdown reduces the specific RNA levels to affect the gene function. RNA digesting enzyme, RNaseA is a very stable and active protein; thus, taking proper precaution and using RNase inactivating solution like RNaseZAP is necessary.

    1. Prepare DEPC treated water for use (see Recipes).

    2. Make sure to use barrier tips, RNase-free microcentrifuge tube (MCT), and glassware.

    3. Put larvae inside a clean RNase-free MCT, flash freeze, and store for RNA isolation.

    4. Dissect sufficient salivary glands from larvae and flash freeze glands inside liquid nitrogen.

    5. Crush the salivary gland/larva using RNase free pestle and follow the manufacturer's protocol for RNA extraction given in the kit.

    6. Crush flash-frozen tissues directly using a pestle or first add lysis solution from the RNA isolation kit and then crush the tissue or larva in the solution.

    7. If adding the lysis solution before crushing, add half of the lysis buffer's final volume, and properly crush the tissue/larva. Subsequently, add the remaining lysis buffer.

    8. Check the purity of RNA using a UV-VIS spectrophotometer/nanodrop. A260/280 and A260/230 ratio equal to 2.0-2.2 suggests pure RNA. Lower values of these ratios indicate the presence of contaminants like proteins, carbohydrates, or phenol.

    9. In an MCT, heat the purified RNA sample at 65 °C for 10 min in a 25% formamide containing RNA loading dye.

    10. Keep RNA sample tubes on ice for 2 min and load immediately on the EtBr containing 1% agarose gel made in Tris-Acetate EDTA (TAE) buffer and run the gel to check the integrity of the isolated RNA.

    11. Prepare cDNA as per instruction given with the BioRad cDNA synthesis kit and store at -20 °C until further use.

    12. A typical cDNA synthesis reaction in 20 µl final volume contains 1 µg purified RNA, 1× iScript reaction mixture, 1 µl of iScript reverse transcriptase enzyme.

    13. Reaction steps include 5 min of priming at 25 °C, 20 min of reverse transcription at 46 °C, and 1 min of inactivation at 95 °C.

    14. Use gene-specific primers (200 nM) on prepared cDNA and run a typical PCR reaction (see Table 1 for primer sequences).

      Note: We used a standard annealing temperature (Ta) of 55 °C for all semiquantitative PCR.

    15. Use RpL49 primers as an internal control gene in the PCR reaction (see Table 1 for primer sequences).


      Table 1. List of primers used for assessing the knockdown levels



    16. Load 50% of PCR product on EtBr containing 1% Agarose gel to assess the extent of knockdown (Figure 4).



      Figure 4. Semiquantitative PCR based assessment of knockdown. cDNAs prepared from Control and nucleoporin knockdown used in a PCR reaction. Upper panels show levels of Nup160 (A), Nup107 (B), and Sec13 (C) from control and knockdown tissues. RpL49 served as an internal loading control between control and knockdown samples.


  3. Dissection and Staining of Salivary gland

    Salivary gland is post-mitotic tissue and carries large nuclei. It possesses polytene chromosomes, which serve as excellent model tissue in several cytological studies and gene expression analyses. The loss of nucleoporins and consequent perturbations in nuclear pore assembly and functions can be studied using immunostaining. dElys was knocked down in the salivary glands using RNAi. Subsequent steps in the dissection of salivary glands in immunostaining are the following.

    1. Dissect out salivary glands in PBS on a silicone elastomer plate from 20 third instar larvae.

      Note: Replace PBS solution after every 2-3 dissections.

    2. Grab the larva approximately at the middle portion of its size using forceps.

    3. Use Dumont 5 forceps to grab the mouth-hook and pull out the head complex.

    4. Simultaneously remove the gut region and other carcass tissues using Dumont 55 forceps.

    5. Use pointed Dumont 5 forceps to grab the head complex by mouth-hook all this while.

    6. Clean the dissected salivary gland by removing the brain, imaginal discs, and fat tissue and ensure that the mouth-hook remains attached.

    7. After dissecting each salivary gland, immediately transfer it to an MCT containing 1 ml of 4% paraformaldehyde.

    8. After all the salivary glands are dissected and collected, keep the MCT on an orbital shaker with gentle agitation for 40 min at room temperature.

      Note: Fixation should not go beyond 1 h; otherwise, the tissue becomes fragile, and staining is affected.

    9. Remove paraformaldehyde and wash tissues three times with PBS-T for 10 min each.

      Note: Do all the wash steps carefully to avoid the loss of salivary glands. It applies to all subsequent steps.

    10. Dissecting the salivary gland with the mouth hook attached to it gives an additional advantage that we can easily see the salivary gland, and thus their loss will be restricted.

    11. Block salivary glands in 3% normal goat serum containing PBS-T or 3% Bovine Serum Albumin (BSA) in PBS-T.

    12. Keep at room temperature for 1 h on an orbital shaker with 110 rotations per minute (rpm).

      Note: Perform all incubations/wash on an orbital shaker set at 110 rpm.

    13. Add 200 µl of anti-dElys and mAb414 primary antibodies containing solution (1:800 dilution of each of the two antibodies prepared in PBS-T).

    14. Incubate overnight at 4 °C on a shaker.

    15. Wash samples three times with 200 µl of PBS-T for 10 min each at room temperature (25 °C).

    16. Add 200 µl of Alexa Fluor-488 or Alexa Fluor-568 conjugated secondary antibodies dilution (1:800 dilution prepared in PBS-T) and incubate at room temperature (25 °C) for 1.5-2.0 h.

    17. Wash samples three times with 200 µl of PBS-T, 10 min each at room temperature (25 °C).

    18. Put 15 µl of Fluoroshield with DAPI mounting medium.

    19. Pick all salivary glands using a cut 200 µl pipette tip.

      Note: Cut the sample inlet end of the fine 200 µl pipette tip to avoid any tissue damage.

    20. Place salivary glands on a previously cleaned slide and spread the tissue gently using Dumont 55 forceps.

      Note: Do not damage the tissue while spreading. Use the mouth hook to grab the tissue, only if required.

    21. Carefully place a clean coverslip on the tissue.

    22. Avoid trapping air bubbles between the slide and coverslip.

    23. Gently remove the excess mounting medium using tissue paper and seal the coverslip edges with transparent nail polish.

    24. Observe processed tissue under a fluorescent Leica microscope.

    25. On Zeiss LSM780, use the control slide to adjust settings and focus in a field with a clear view of cells.

    26. Scan the field having salivary gland nuclei under 63× objective in a Confocal Laser Scanning Microscope LSM780.

    27. Capture images from different visual fields of the control slide (Figure 5).

    28. Reuse the same settings for the knockdown samples also.



      Figure 5. RNAi mediated dElys knockdown depleted dElys signals from salivary gland nuclei. The salivary glands dissected from Control RNAi (upper panels) and dElys RNAi (lower panels) third instar larvae and stained with dElys (red, first panels), mAb414 (green, second panels), DAPI (blue, third panels). The fourth panels represent the merged images. Scale bars =10 μm.


  4. Wing and Eye specific knockdown

    1. Set up crosses between nup160 RNAi and +/+; Ey-GAL4/Ey-GAL4; UAS-Dicer/UAS-Dicer (for eye-specific knockdown) or nup160 RNAi and +/+; wg-GAL4/Wg-GAL4; UAS-Dicer/UAS-Dicer (for wing-specific knockdown).

    2. Transfer crosses to a lower temperature (18 °C). The lower temperature reduces the GAL4 activity.

    3. Reduced GAL4 activity at 18 °C causes a reduction in knockdown levels and allows adult fly emergence.


  5. Dissection and visualization of eyes and wings

    1. Visualization of eyes

      Drosophila compound eyes are complex yet pertinent tissue to observe growth defects upon RNAi mediated knockdown. A definite transcriptional paradigm regulates the arrangement of each ommatidium in the compound eye during development. Any perturbation in the ommatidium arrangement, their smooth morphology, and the presence of a bristle with each ommatidium due to RNAi mediated knockdown serves to indicate the importance of the gene function. After knocking down the Nup160 using RNAi, process wings in the following manner. If the gene of interest induces embryonic to larval lethality, eye-specific knockdown helps assess the gene function. After knocking down the Nup160 using RNAi, process compound eyes in the following manner.

      1. Collect adult flies in an empty vial.

      2. Apply 100-200 µl of diethyl ether on cotton and plug the fly containing vial to anesthetize them.

      3. Orient them properly (compound eye facing up) under Stereomicroscope Leica S6E.

      4. Fix the position of the fly using carbon conductive tape for steady observation of eyes.

      5. Observe eye morphology using Leica fluorescent stereomicroscope M205 FA at ×123 magnification (left panels in Figure 6A).

    2. SEM analysis of eye phenotype

      1. Anesthetize the flies using diethyl ether. Flash freeze them and separate heads from the body.

      2. Fix head tissues with 2.5% Glutaraldehyde in PBS for 2 h at 4 °C.

      3. Wash with PBS and 4% sucrose solution.

      4. Dehydrate tissue using a series of graded ethanol wash (single wash with 25%, 50%, 75% ethanol, and twice with 100% ethanol) for 2 h each at room temperature.

      5. Keep inside a lab desiccator for critical point drying.

      6. Mount dried tissue on aluminum stubs with carbon-conductive tape.

      7. Coat the flies with gold particles in a sputter coating apparatus.

      8. Image using Zeiss Gemini II FESEM with In lens detector (middle and right panels in Figure 6A).

    3. Visualization of wings

      Drosophila adult wings serve as an excellent tissue to observe the developmental defects induced by perturbation in a gene function. The changes in bristles, wing veins, and notched wings are distinct phenotypes seen with the knockdown of genes. Wing imaginal discs are present in the third instar larva. If the gene of interest induces post-larval lethality, wing imaginal discs are routinely analyzed. After knocking down the Nup160 using RNAi, process wings for visualization in the following manner.

      1. Follow Steps E1a and E1b of the visualization of eyes procedure.

      2. Cut the wings with scissors and place them on a clean slide.

      3. Put coverslip on top of the wing tissue and seal.

      4. Observe under upright light microscope Leica DM2500 at 10× magnification (panels in Figure 6B).



    Figure 6. Knockdown of Nup160 in eyes and wings induces developmental defects. (A) Eye specific knockdown of Nup160 using Ey-GAL4 and visualization of the compound eye under bright field light microscope and SEM. (B) Wing specific knockdown of Nup160 using Wg-GAL4 and visualization of wings under bright field light microscope. The control knockdown is for comparison. Scale bars (A) are 200 μm in the left panels, 20 μm in the middle panels, and 2 μm in the right panels. The scale bar in (B) is 200 μm.

Data analysis

We have utilized RNAi mediated knockdown of dElys in Drosophila and have explored the functional significance of dElys in Drosophila development. Observations made regarding dElys function highlight that dElys is important for embryonic development, nuclear pore assembly, nucleocytoplasmic shuttling of the developmentally regulated molecule, Dorsal. The genetic crosses between flies carrying RNAi against dElys, Nup160, Nup107, and Sec13, and promoter-specific GAL4 sequences produced desired progenies where both RNAi and UAS-GAL4 were present in the same fly. Progenies were selected at the third instar larva stage based on the absence of GFP expression (non-GFP larva). In salivary gland specific analysis, we dissected paired salivary glands from non-GFP larvae (Figure 3). Use of Actin5C-GAL4 for knockdown of genes of interest, Nup160, Nup107, and Sec13 successfully achieved significant gene expression knockdown. The semiquantitative RT-PCR analysis established that these genes are efficiently depleted (Figure 4).

    We achieved a knockdown ranging from 50-80% using RNAi for different genes. Determination of RpL49 gene levels by PCR helped in making these precise calculations. In conditions where the ubiquitous knockdown appears lethal or harmful, we explored the option of tissue-specific knockdown. Additionally, when a gene's function confines to a particular tissue, the power of tissue-specific promoter mediated knockdown can be utilized. Any potential off-target gene of utilized RNAi must also be analyzed to build a correlation between gene and phenotype. Mehta et al., 2020 reported that two off-targets of dElys RNAi were not perturbed significantly. Simultaneously, a gentle lowering of RNA transcript for a dosage-sensitive protein can also bring about the observable phenotypes.

    dElys levels reduced strongly under dElys knockdown conditions (Actin5C>dElys). The ubiquitous knockdown of dElys and subsequent analysis of the salivary gland nuclei indicated that nuclear pore assembly is affected by dElys depletion. Under similar conditions, dElys levels are unperturbed in control knockdown (Actin5C>Control) (Figure 5). mAb414 antibody recognizes four distinct FG-repeat rich nucleoporins. A noticeable reduction in mAb414 staining of nuclear pores is also evident upon dElys RNAi. Based on these observations, we suggest an essential role for dElys in nuclear pore assembly in Drosophila.

    We further utilized the UAS-GAL4 system's power for RNAi mediated knockdown of Nup160 in Drosophila eye and wings. Eye-specific Ey-GAL4 and wing-specific wg-GAL4 promoters depleted Nup160 to produce significant perturbation in the development of these two tissues. In Nup160 knockdown animals, the entire compound eye is shrunken and seems absent. The SEM imaging of knockdown eyes shows drastic changes compared with eye-specific control knockdown (Figure 6, panel A). Similarly, the wing-specific knockdown of Nup160 caused a reduction in the wing blade size, and the veins are completely missing (Figure 6, panel B).

    The observations made with RNAi mediated knockdown of nucleoporin genes dElys and Nup160 established the robustness of this gene knockdown paradigm. Further, the tissue-specific knockdown and analysis from salivary glands, eyes, and wings highlighted the functional significance of nucleoporins in Drosophila development.

Recipes

  1. Phosphate Buffer Saline (1×)

    Sodium Chloride 137 mM

    Potassium Chloride 2.7 mM

    di-Sodium hydrogen phosphate heptahydrate 10 mM

    Potassium dihydrogen phosphate 1.8 mM

  2. PBS-T

    Lukewarm the falcon tube containing 1× PBS and add Triton X-100 (100%) to a final concentration of 0.2% with a cut pipette tip.

  3. Fly food ingredients

    Ingredient                              Amount/liter

    Corn flour                               80 g

    Sugar                                       40 g

    Dextrose                                 20 g

    Yeast Extract                          15 g

    Agar                                         10 g

    Methyl-4-hydroxybenzoate 1 g

    Propionic acid                        4 ml

    Orthophosphoric acid          0.6 ml

    Ethanol absolute                      10 ml

    1. Weigh corn flour, sugar, dextrose, yeast extract, Agar separately, and add to the flask.

      Note: Follow the ratio of different components mentioned in the table above.

    2. Autoclave at 121 °C for 20 min and let it cool for some time till the temperature reaches ~50-55 °C.

    3. Meanwhile, weigh 1 g of methyl-4-hydroxybenzoate and dissolve in 10 ml ethanol.

    4. Once the medium has cooled down to ~50-55 °C (check it with a thermometer), add methyl-4-hydroxybenzoate solution, propionic acid, and orthophosphoric acid to the media.

    5. Mix it thoroughly and pour 8-10 ml into each glass vial.

      Note: Avoid the incorporation of bubbles. Do not let it cool below ~50 °C; otherwise, Agar will start to solidify.

    6. Once the media has solidified inside the vial, put a tight cotton plug on each vial, and use it as required.

      Note: Keep vials inside a cage for cooling. The cage helps avoid contamination of vials by stray flies in the room.

  4. RNA loading dye

    50 µl Formamide

    35 µl 6× DNA loading dye

    15 µl Ultrapure water (to make up the final volume)

  5. DEPC treated water

    1. Add 1 ml of DEPC to 1,000 ml of ultrapure water (0.1%).

    2. Mix well, cover the bottle and leave it overnight at room temperature.

    3. Autoclave two times, let it cool down to room temperature.

    4. Make aliquots and store them at -20 °C for use.

  6. TAE Buffer (50×)

    1. Add 242 g of Tris base to 700 ml of double-distilled water.

    2. Add 57.1 ml of Glacial acetic acid carefully.

    3. Mix 100 ml of 0.5 M EDTA having pH 8.0 to it.

    4. Adjust volume to 1,000 ml.

      Note: The pH should be around 8.5 and need not be adjusted.

    5. Use 20 ml of 50× TAE stock to make a 1 liter of 1× TAE buffer.

    6. 1× TAE buffer will have the following final concentrations: 40 mM Tris base, 20 mM Acetic acid, and 1 mM EDTA

  7. 4% Paraformaldehyde

    1. Take 50 ml of 1× PBS in a clean glass bottle.

    2. Add 4 g of paraformaldehyde while keeping the bottle on the heating plate at ~60 °C

      Note: Make sure that to work inside a ventilated hood as paraformaldehyde is hazardous.

    3. Add 1 N NaOH slowly and dropwise to dissolve all the paraformaldehyde.

    4. When all paraformaldehyde dissolves in PBS, adjust the pH to 7.2-7.4 using 1 N HCl.

    5. Adjust the final volume to 100 ml using 1× PBS.

    6. Filter and store paraformaldehyde as 10 ml aliquots at 0-4 °C.

      Note: Do not store the paraformaldehyde solution for more than a month.

Acknowledgments

This protocol is originally published in a research manuscript by Mehta et al. (2020) and was supported by the Science and Engineering Research Board (SERB, EMR/2016/001819). We thank Ms. Jyotsna Kawadkar for the accurate illustration of the UAS-GAL4 system in Drosophila. We also thank IISER Bhopal, the Fly facility at IISER Bhopal, and DST-FIST Live cell imaging and SEM facilities at IISER.

Competing interests

No competing interest to declare.

Ethics

No human subjects or mouse model was used in the study.

For rearing, maintaining, and experimenting with Drosophila, work followed the applicable parts of animal Ethics guidelines of the host Institute.

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. Mehta, S. J. K., Kumar, V. and Mishra, R. K. (2020). Drosophila ELYS regulates Dorsal dynamics during development. J Biol Chem 295(8): 2421-2437.
  3. 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.

简介

[摘要]功能丧失的研究为基因或基因产物在不同细胞过程中的参与提供了重要启示。短发夹RNA(shRNA)介导的RNA干扰(RNAi)是一种经典而直接的技术,经常用于敲低基因以评估其功能。也可以通过siRNA,microRNA或CRISPR-Cas9方法实现类似的基因表达扰动。在果蝇遗传学中,UAS-GAL4系统用于表达RNAi,并使遍在和组织特异性的基因敲除成为可能。UAS-GAL4系统借鉴了酿酒酵母的遗传成分,因此排除了系统意外表达的可能性。特别地,该技术使用靶标特异性shRNA,并且其表达受上游激活序列(UAS)支配。由特定启动子调节的GAL4受控表达可以普遍或以组织特异性方式驱动干扰RNA的表达。通过RNA分离和半定量RT-PCR反应,然后进行琼脂糖凝胶电泳来测量敲低效率。我们还采用了免疫染色程序来评估击倒效率。

RNAi为研究人员提供了降低基因产物水平(相当于亚同型条件)并研究结果的选择。基于UAS-GAL4的RNAi方法提供了基因表达的时空调节,还有助于推断早期发育阶段所需的基因功能。


[背景]果蝇果蝇(果蝇)是在研究实验室经常使用的一种通用模式生物。果蝇易于处理,繁殖和维护。而且,精心制作却寿命短,繁殖力高的果蝇具有更多的优势。果蝇遗传学工具的易用性有助于发展对基因功能的全面了解。由于果蝇基因中有60%与人类基因同源,并且具有前面提到的其他优点,因此果蝇是研究体内基因功能的显而易见的模型生物。

UAS-GAL4系统用于基因转录的体内激活。此外,有人建议可以使用UAS-GAL4系统表达反义RNA,以实现对基因表达的显着抑制(Brand和Perrimon,1993)。为了在果蝇中使用UAS-GAL4系统,产生了携带能够调节RNAi表达的上游激活序列(UAS)的转基因蝇系。产生了基因组规模的shRNA依赖性RNAi资源,用于果蝇基因的阶段特异性有效敲除(Ni等,2011)。RNAi品系可从维也纳果蝇资源中心(VDRC,维护GD和KK RNAi品系)和果蝇RNAi筛选中心(TRiP和shRNA品系由DRSC生成,目前正在维护,可从Bloomington Drosophila储备中心,BDSC获得)。可以在VDRC库存中心(https://stockcenter.vdrc.at/control/main)和DRSC(https://fgr.hms.harvard.edu/)网站上找到生成不同RNAi品系的详细方法。。

当RNAi飞行系与GAL4驱动飞行系交叉时,后代可以表达UAS-GAL4系统的两个组件。上游启动子指导GAL4表达,无论在何处表达GAL4,它都以高亲和力与UAS序列结合,从而表达RNAi组分(图1)。在缺少GAL4表达的情况下,由于缺乏dsRNA表达,转基因RNAi蝇的行为类似于野生型。该方法允许以时空方式敲低所需基因。经典地,X射线诱变和P元素动员已用于实现基因表达的损失。此外,EMS诱变筛选已用于诱导点突变,偶尔导致影响基因表达的截短。RNAi介导的敲除相当容易且劳动强度较低。





图1.详细说明果蝇中UAS-GAL4系统功能的示意图



由于不同组织中GAL4的表达程度不同,因此在不同组织中可以看到敲低的梯度。此外,GAL4功能的温度依赖性调节提供了调节敲低水平的优势。总之,使用UAS-GAL4系统进行基因表达调控的这些方法可提供有关我们感兴趣基因功能的更多信息。重要的是,该方法提供了对RNAi介导的敲除的受控时空调节,可以帮助研究发育必需基因的功能,具有管家功能的基因以及与有害后果相关的基因突变。该方法还有助于新基因如dElys的功能表征(Mehta等,2020)。

典型的qRT-PCR或半定量PCR验证了RNAi介导的敲低后基因表达水平的降低。可以通过免疫染色和蛋白质印迹法检测所需的基因产物水平来进一步评估击倒效率。我们已经利用唾液腺中RNAi介导的敲除,并通过免疫染色或通过半定量PCR评估mRNA水平来评估其有效性。我们使用RNAi介导的敲除方法成功消除了眼和翅膀中的核孔蛋白,并报道了核孔蛋白在组织和生物体发育中的重要性。

关键字:dElys, 核孔蛋白, RNA干扰, UAS-Gal4, 果蝇, 半定量PCR, 核孔

材料和试剂
1.5 ml微量离心管(Genaxy,目录号:GEN-MT-150-CS)
屏障提示
0.5-10 µl吸头(Axygen Scientific,目录号:TF-300-LRS)
1-20 µl吸头(Axygen Scientific,目录号:TF-20-LRS)
1-200 µl吸头(Axygen Scientific,目录号:TF-200-LRS)
100-1,000 µl吸头(Axygen Scientific,目录号:TF-1000-LRS)
玻璃小瓶和瓶子
SYLGARD 184硅弹性体(DOW ,目录号:1673921)
微量移液器吸头(2、20、200、1,000 µl)
0.5-10 µl吸头(Axygen Scientific,目录号:T-300-LRS)
1-200 µl吸头(Axygen Scientific,目录号:TR-222-CLRS)
100-1,000 µl吸头(Axygen Scientific,目录号:T-1000-CLRS)
3厘米皿(Eppendorf,货号:0030700112)
食品瓶
CO 2垫
棉塞
玉米粉
蔗糖
酵母提取物(HiMedia,目录号:RM027)
琼脂(Merck,目录号:A5306)
葡萄糖(HiMedia,目录号:GRM077)
4-羟基苯甲酸甲酯(Sigma-Aldrich,目录号:H5501)
正磷酸(默克,目录号:100573)
丙酸(HiMedia,目录号:GRM3658)
乙醇(常熟市宏盛精细化工CHN01)
液氮
RNA分离试剂盒(Favorgen,目录号:FATRK001)
RNaseZAP(Thermo Scientific,目录号:AM9780)
DNase(Thermo Scientific,目录号:EN0523)
焦碳酸二乙酯(HiMedia,目录号:MB076)
乙醇(MP Biomedicals,目录号:180077)
甲酰胺(Sigma-Aldrich,目录号:F7503)
6×DNA加载染料紫色(新英格兰生物实验室,目录号:B7024S)
cDNA合成试剂盒(iScript,Bio-Rad ,目录号:1708891)
琼脂糖(Invitrogen,货号:16500500)
溴化乙锭(EtBr)(MP Biomedicals,目录号:193993)
定制引物(整合DNA技术)
G9-Taq DNA聚合酶(GCC Biotech,目录号:G7115A)
dNTPs解决方案组合(新英格兰生物实验室,目录号:N0447L)
氯化钠(Sigma-Aldrich,目录号:S3014)
氯化钾(Sigma-Aldrich,目录号:P5405)
七水合磷酸氢二钠(Merck,目录号:1065751000)
磷酸二氢钾(Sigma-Aldrich,目录号:P9791)
Tris-base(Sigma-Aldrich,目录号:T6066)
冰醋酸(Merck EMPLURA,目录号:1.93402.2521)
碳导电胶带(TED PELLLA Inc.目录号:16073-1)
铝制短管(TED PELLLA Inc.目录号:16111-9)
戊二醛(Sigma-Aldrich,目录号:G5882)
金靶(Quorum Technology inc,目录号:SC502-314A)
EDTA(Merck EMPARTA,货号:1.93312.1021)
多聚甲醛(Sigma-Aldrich,目录号:158127)
海卫一X-100(Sigma-Aldrich,目录号:X100)
载玻片(Borosil,目录号:9100P02)
盖玻片(Borosil,目录号:9115S01)
牛血清白蛋白(默克,目录号:621650500501730)
带有DAPI的氟防护板(Sigma-Aldrich,目录号:F6057)
mAb414(Biolegend 902901)-mAb414识别富含FG的核孔的四个不同核孔蛋白
甲nti- d ELYS(梅塔等人,2020)
Alexa Fluor-488(Invitrogen,目录号:A-11029)
Alexa Fluor-568(Invitrogen,目录号:A-11036)
Coverslip密封胶(透明指甲油)
乙醚(Merck,目录号:107026)
磷酸盐缓冲盐水(1×)(请参阅食谱)
PBS-T(请参阅食谱)
飞食品成分(请参阅食谱)
RNA加载染料(请参见食谱)
经DEPC处理的水(请参阅配方)
TAE缓冲液(50×)(请参阅配方)
4%多聚甲醛(请参阅食谱)

飞股票

控制野生型(w 1118 )

驱动线(内部生成的组合)

+ / +; 肌动蛋白5C-GAL4 / CyO-GFP; UAS-Dicer / UAS-Dicer
+ / +; wingless -GAL4 / wingless -GAL4; UAS-Dicer / UAS-Dicer
+ / +; 眼睛-GAL4 /眼睛-GAL4; UAS-Dicer / UAS-Dicer
RNAi系

+ / +; v103547 / v103547;UAS-Dicer / UAS-Dicer(内部生成的dElys RNAi KK系列与UAS-Dicer结合使用)
dElys RNAi KK系列(VDRC,VDRC ID:103547)
Nup160 RNAi GD系列(VDRC,VDRC ID:21937)
Sec13 RNAi KK系列(VDRC,VDRC ID:110428)
Nup107 RNAi GD系列(VDRC,VDRC ID:22407)

设备


微量移液器(Nichiriyo,Gilson,康宁)
BOD孵化器
密理博净水器
Genova Nanodrop(Bibby Scientific,型号:737501)
镊子Dumont 5(精细科学工具,目录号:11295-10)
镊子Dumont 55(精细科学工具,目录号:11295-51)
剪刀(精细科学工具,目录号:91500-09)
徕卡DM2500
荧光立体显微镜(Leica,型号:M205 FA)
共焦激光扫描显微镜(蔡司,型号:LSM780)
体视显微镜(徕卡,型号:S6E)
扫描电子显微镜(蔡司,型号:Gemini II Ultra plus)
台式离心机(Eppendorf,型号:5424,Thermo Scientific MicroCL 21R)
PCR机(Applied Biosystems,型号:2720热循环仪)
水浴(Grant- B io,型号:PSU-10i)
凝胶电泳仪(CBS小号系统求解,型号:GCMGU-202T)
电源组(CBS小号系统求解,型号:EPS 300)
UVP MultiDoc-It数字成像系统(UVP,目录号:97-0192-02)

软件


ZEN 3.2蓝色版(Zeiss)
ImageJ /斐济(免费,NIH)

程序


果蝇中UAS-GAL4依赖性和RNAi介导的基因敲低需要几个步骤。它包括以下步骤:饲养生物,建立遗传杂交,收获所需遗传组合的幼虫,RNA分离和敲除评估,解剖,免疫染色和表型分析。下面提到的是组织制备和处理的一般方案(图2)。





图2.包含协议细节的流程图


RNAi介导的基因敲除的遗传杂交
成长+ / +; 肌动蛋白5C-GAL4 / CyO-GFP; UAS-Dicer / UAS-Dicer在装有飞行培养基的小瓶中大量飞行。
将具有GAL4驱动程序的原始雌性收集在单独的培养基瓶中。
生长RNAi和野生型(w 1118 )在不同的培养基小瓶中飞行,以获得足够的雄性苍蝇。
将6-7个原始年龄的雌性雌性和2-3个相同年龄(3-4天大)的UAS-RNAi雄性放入一个新鲜的培养基小瓶中,以建立交配杂交。
对于对照杂交,使用w 1118雄性代替UAS-RNAi雄性,并按照步骤A4的要求将原生雌性杂交。
将小瓶与蝇一起在室温下孵育24小时,以进行遗传杂交。 
根据实验设计,将其移至28°C,25°C或18°C。
注意:在较高温度下温育可使GAL4活性更高,从而诱导RNAi表达增加。

将杂交物放在设定温度的培养箱中,直到看到第三龄幼虫。 
取出成年苍蝇,放入2-3 ml水或2%蔗糖溶液,从培养基的顶层收获幼虫。
注意:步骤A8至A13在图3中。

使用画笔将食物和水的上层混合,然后将其收集在干净的塑料皿中。
在干净的装有蒸馏水/磷酸盐缓冲液的平板中分离第三龄幼虫。
用水清洗收集的幼虫2-3次以清洁它们。
从GFP(+ / +; RNAi / CyoGFP; UAS-Dicer / +)幼虫中分离出非GFP幼虫(+ / +;肌动蛋白5C / RNAi; UAS-Dicer / +,表明所需的驱动基因和RNAi遗传组合)后续处理。
对于对照,按照步骤A5所述,使用来自对照杂交的非GFP幼虫。
从PBS中非GFP第三龄幼虫的唾液腺或肌动蛋白5C-GAL4驱动的RNAi的头部复合物中分离出来。
注意:有关唾液腺解剖的详细信息,请参见下面的图3和C部分中提到的步骤1-5。





图3.从果蝇三龄幼虫中分离唾液腺的步骤。上面板(af),幼虫分离和清洁步骤;(a)食物小瓶中的幼虫和P,(b)喷水,并用刷子将食物和幼虫的顶层悬浮,(c)将培养皿中的悬浮液倾析,(d)将食物和三龄幼虫悬浮, (e)用蒸馏水收集的清洁幼虫,(f)第三龄幼虫。下面板(gl)幼虫解剖和唾液腺分离的步骤(g)第三龄幼虫,(h)用镊子握住第三龄幼虫,(i)拔出唾液腺和第三龄幼虫的腹部,(j )头部复合体,具有大脑,椎间盘和唾液腺(k)带有相关脂肪体的唾液腺,(l)解剖并清理了一对附有口钩的唾液腺。


RNA分离
该方法最关键的步骤是RNA分离。RNAi介导的敲低降低了特定的RNA水平,从而影响基因功能。RNA消化酶,RNaseA是一种非常稳定和活跃的蛋白质。因此,必须采取适当的预防措施并使用RNaseZAP之类的RNase灭活溶液。

准备使用DEPC处理过的水(请参阅配方)。
确保使用隔离针尖,无RNase的微量离心管(MCT)和玻璃器皿。
将幼虫放入干净的无RNase的MCT中,快速冷冻,并保存以进行RNA分离。
从幼虫解剖足够的唾液腺,并在液氮中快速冷冻。
使用不含RNase的杵粉碎唾液腺/幼虫,并按照试剂盒中提供的RNA提取方法进行操作。
使用杵直接粉碎速冻组织,或先添加RNA分离试剂盒中的裂解液,然后将溶液中的组织或幼虫粉碎。
如果在压碎前添加裂解液,则添加一半的裂解缓冲液最终体积,并适当压碎组织/幼虫。随后,添加剩余的裂解缓冲液。
使用UV-VIS分光光度计/ nanodrop检查RNA的纯度。甲260/280和甲230分之260比等于2.0-2.2表明纯的RNA。这些比率的较低值表示存在蛋白质,碳水化合物或苯酚等污染物。
在MCT中,将纯化的RNA样品在含有25%甲酰胺的RNA负载染料中于65°C加热10分钟。
将RNA样品管在冰上放置2分钟,然后立即加载到Tris-醋酸EDTA(TAE)缓冲液中制成的含1%琼脂糖凝胶的EtBr中,然后运行凝胶以检查分离的RNA的完整性。
按照BioRad cDNA合成试剂盒中的说明制备cDNA,并保存在-20°C下直至进一步使用。
最终体积为20 µl的典型cDNA合成反应包含1 µg纯化的RNA,1x iScript反应混合物,1 µl iScript逆转录酶。
反应步骤包括在25°C进行5分钟的引发,在46°C进行20分钟的逆转录和在95°C进行1分钟的灭活。
在准备的cDNA上使用基因特异性引物(200 nM)并进行典型的PCR反应(引物序列请参见表1)。
注意:对于所有半定量PCR,我们使用的标准退火温度(Ta)为55°C。

在PCR反应中使用RpL49引物作为内部控制基因(引物序列请参见表1 )。

表1.用于评估组合水平的引物列表

Sl#

底漆名称

顺序(5' → 3')

1个

Nup107F

CTGGGAGCACAAGGTTAAGG

2

                            Nup107R

GATCTCAGGAATGCAGATGGAG

3

Nup160F

CCAGTGGCTGATAAACTCCTAC

4

Nup160R

AGAGCTGTGGGTTTGCTATG

5

Sec13F

TCACATTGTGGAAGGAGAACAC

6

                            Sec13R

GTTGGACAGGTTCGAGCTATG

7

RpL49F

CGTTTACTGCGGCGAGAT

8

RpL49R

GTGTATTCCGACCACGTTACA


在含1%琼脂糖凝胶的EtBr上加载50%PCR产物以评估敲除程度(图4)。



图4.基于半定量PCR的击倒评估。由对照和核孔蛋白敲低制备的cDNA用于PCR反应。上图显示了来自对照和基因敲除组织的Nup160 (A),Nup107 (B)和Sec13 (C)的水平。RpL49用作对照样品和组合样品之间的内部装载对照。


唾液腺的解剖和染色
唾液腺是有丝分裂后的组织,带有大核。它具有多聚体染色体,在许多细胞学研究和基因表达分析中可作为优秀的模型组织。可以使用免疫染色研究核孔蛋白的损失以及随之而来的核孔装配和功能的扰动。dElys使用RNAi在唾液腺中被击倒。免疫染色中唾液腺解剖的后续步骤如下。

在20只三龄幼虫的硅酮弹性体板上的PBS中解剖唾液腺。
注意:每隔2-3个解剖后更换PBS溶液。

使用镊子将幼虫大约抓住其大小的中间部分。
使用Dumont 5镊子抓住口钩并拔出头部。
同时使用Dumont 55镊子去除肠道区域和其他car体组织。
在这段时间内,使用尖头的Dumont 5镊子通过嘴钩抓住头部。
去除大脑,假体椎间盘和脂肪组织,以清洁解剖的唾液腺,并确保口钩保持附着状态。
解剖每个唾液腺后,立即将其转移至含有1 ml 4%多聚甲醛的MCT中。
解剖并收集所有唾液腺后,将MCT放在室温摇动的摇床上振动40分钟。
注意:固定时间不得超过1小时;否则,组织将变得脆弱,并影响染色。

去除多聚甲醛并用PBS-T清洗组织3次,每次10分钟。
注意:仔细进行所有清洗步骤,以免流涎腺丢失。它适用于所有后续步骤。

用连接的钩子解剖唾液腺还有一个额外的优点,就是我们可以很容易地看到唾液腺,因此可以减少唾液腺的损失。
在含PBS-T的3%正常山羊血清或PBS-T中的3%牛血清白蛋白(BSA)中阻断唾液腺。
在轨道振动器上以110转/分钟(rpm)在室温下保持1小时。
注意:在设定为110 rpm的定轨振荡器上进行所有孵育/洗涤。

加入200 µl含溶液的抗d Elys和mAb414一抗(在PBS-T中制备的两种抗体中的每一种的1:800稀释度)。
在振荡器上于4°C孵育过夜。
在室温(25°C)下,用200 µl PBS-T洗涤样品3次,每次10分钟。
加入200 µl Alexa Fluor-488或Alexa Fluor-568缀合的二抗稀释液(在PBS-T中以1:800稀释液稀释),然后在室温(25°C)下孵育1.5-2.0 h。
用200 µl PBS-T洗涤样品3次,在室温(25°C)下每次10分钟。
放入含DAPI安装介质的15 µl Fluoroshield。
使用切好的200 µl移液器吸头挑选所有唾液腺。
注意:切下200 µl细吸管吸头的样品入口端,以避免任何组织损伤。

将唾液腺放在先前清洁过的载玻片上,并用Dumont 55镊子轻轻地铺开组织。
注意:散布时请勿损坏组织。仅在需要时,使用口钩抓住组织。

将清洁的盖玻片小心地放在薄纸上。
避免在载玻片和盖玻片之间夹带气泡。
用薄纸轻轻去除多余的安装介质,并用透明指甲油密封盖玻片的边缘。
在荧光徕卡显微镜下观察处理过的组织。
在Zeiss LSM780上,使用控制滑块调整设置并聚焦在具有清晰单元格视图的字段中。
用共聚焦激光扫描显微镜LSM780在63 ×物镜下扫描具有唾液腺核的视野。
从控制幻灯片的不同视野捕获图像(图5)。
还可以对击倒样本重新使用相同的设置。



图5. RNAi介导的唾液腺核中的dElys击倒耗尽的d Elys信号。唾液腺从对照RNAi(上图)和dElys RNAi(下图)第三龄幼虫中解剖,并用d Elys(红色,第一图),mAb414(绿色,第二图),DAPI(蓝色,第三图)染色。第四个面板代表合并的图像。比例尺= 10μm。


机翼和眼睛特定的组合
在nup160 RNAi和+ / +之间建立杂交;Ey -GAL4 / Ey -GAL4; UAS-Dicer / UAS-Dicer(用于眼睛特异性敲除)或nup160 RNAi和+ / +;wg -GAL4 / Wg -GAL4; UAS-Dicer / UAS-Dicer(用于机翼特定的组合)。
转移至较低温度(18°C)。较低的温度降低了GAL4的活性。
GAL4活性在18°C时降低,导致击倒水平降低,并允许成年苍蝇出现。

解剖和可视化眼睛和翅膀
眼睛的可视化
果蝇复眼是复杂但相关的组织,可观察到RNAi介导的敲低后的生长缺陷。确定的转录范例在发育过程中调节复眼中每个邻眼的排列。由于RNAi介导的敲除,小孔排列中的任何扰动,其平滑的形态以及每个小孔均存在刷毛的现象表明了基因功能的重要性。使用RNAi敲除Nup160后,以以下方式处理机翼。如果目的基因诱导了幼虫的致死性,那么眼睛特异性的敲低有助于评估基因的功能。使用RNAi敲除Nup160后,按以下方式处理复眼。

将成年苍蝇收集在一个空的小瓶中。
在棉花上涂抹100-200 µl乙醚,然后塞上装有小瓶的果蝇以麻醉它们。
在立体显微镜Leica S6E下正确对准它们(复眼朝上)。
使用碳导电带固定蝇的位置,以稳定观察眼睛。
使用Leica荧光立体显微镜M205 FA以123倍的放大倍率观察眼睛的形态(图6A的左图)。
眼表型的SEM分析
用乙醚麻醉果蝇。闪光灯会冻结它们,并使头部与身体分开。
在4°C下用2.5%戊二醛的PBS固定头部组织2 h。
用PBS和4%蔗糖溶液洗涤。
在室温下,使用一系列分级的乙醇洗涤液(分别用25%,50%,75%乙醇洗涤一次,并用100%乙醇洗涤两次)使组织脱水。
保留在实验室干燥器中以进行临界点干燥。
用碳带将干燥的纸巾固定在铝制的短管上。
在溅射镀膜设备中用金颗粒覆盖苍蝇。
使用带In镜头检测器的Zeiss Gemini II FESEM拍摄的图像(图6A中的中间和右侧面板)。
机翼的可视化
果蝇的成年翅是观察基因功能微扰引起的发育缺陷的优良组织。刚毛,翼状静脉和有缺口的翼的变化是基因敲除后所见的独特表型。机翼想象中的盘存在于三龄幼虫中。如果感兴趣的基因引起幼虫后致死,则常规分析机翼假想盘。使用RNAi敲除Nup160后,以以下方式处理翅膀以进行可视化。

遵循眼睛可视化过程的步骤E1a和E1b。
用剪刀剪掉机翼,然后将它们放在干净的幻灯片上。
将盖玻片放在机翼组织的顶部并密封。
在直立的光学显微镜Leica DM2500下以10倍的放大倍率观察(图6B中的面板)。



图6.击倒眼睛和翅膀中的Nup160会导致发育缺陷。(A)使用Ey- GAL4对Nup160的眼睛特异性敲除,并在明场光学显微镜和SEM下可视化复眼。(B)使用Wg- GAL4对Nup160进行机翼特异性敲低并在明场光学显微镜下观察机翼。控件组合键用于比较。比例尺(A)在左侧面板中为200μm,在中间面板中为20μm,在右侧面板中为2μm。(B)中的比例尺为200μm。


数据分析


我们利用RNA干扰介导击倒的dElys在果蝇和已经探索的功能意义d ELYS在果蝇的发展。关于dElys功能的观察结果表明,d Elys对于胚胎发育,核孔装配,发育调控分子Dorsal的核质穿梭非常重要。携带针对dElys ,Nup160 ,Nup107和Sec13的RNAi的果蝇之间的遗传杂交,以及启动子特异的GAL4序列产生了所需的后代,其中同一蝇中同时存在RNAi和UAS-GAL4。根据缺少GFP表达的昆虫(非GFP幼虫),在第三龄幼虫阶段选择后代。在唾液腺特异性分析中,我们从非GFP幼虫中解剖了成对的唾液腺(图3)。使用肌动蛋白5C-GAL4敲低感兴趣的基因Nup160 ,Nup107和Sec13可以成功实现显着的基因表达敲低。半定量RT-PCR分析确定这些基因被有效地消耗掉了(图4)。
使用不同基因的RNAi,我们获得了50-80%的敲低。通过PCR确定RpL49基因水平有助于进行这些精确的计算。在普遍存在的击倒表现为致命或有害的情况下,我们探索了组织特异性击倒的选择。另外,当基因的功能仅限于特定组织时,可以利用组织特异性启动子介导的敲低的能力。还必须对利用的RNAi的任何潜在脱靶基因进行分析,以建立基因与表型之间的相关性。Mehta等。,2020年报道,dElys RNAi的两个脱靶点并未受到明显干扰。同时,剂量敏感性蛋白的RNA转录物温和降低也可产生可观察到的表型。
在敲除dElys的条件下,dElys水平会大大降低(肌动蛋白5C> dElys )。dElys普遍存在的敲低和唾液腺核的后续分析表明,d Elys耗竭会影响核孔的组装。在相似条件下,对照敲低中的dElys水平不受干扰(肌动蛋白5C>对照)(图5)。mAb414抗体识别四种不同的FG重复富核孔蛋白。在dElys RNAi上,mAb414核孔染色也明显减少。根据这些观察结果,我们建议d Elys在果蝇的核孔装配中起重要作用。

我们进一步利用UAS-GAL4系统的电源的RNAi介导的击倒的Nup160在果蝇眼睛和翅膀。眼睛特异性Ey -GAL4和翅膀特异性wg -GAL4启动子耗尽了Nup160,从而在这两个组织的发育中产生了明显的干扰。在Nup160组合式动物中,整个复眼都缩小了,似乎没有了。与特定于眼球的对照击倒相比,击倒眼的SEM图像显示出急剧的变化(图6,图A)。类似地,Nup160的特定于机翼的击倒导致机翼叶片尺寸减小,并且静脉完全缺失(图6,B板)。
RNAi介导的核孔蛋白基因dElys和Nup160的敲除的观察结果确定了该基因敲除范例的鲁棒性。此外,唾液腺,眼睛和翅膀的组织特异性敲除和分析突出了核孔蛋白在果蝇发育中的功能意义。


菜谱


磷酸盐缓冲盐水(1×)
氯化钠137 mM

氯化钾2.7 mM

七水合磷酸氢二钠10 mM

磷酸二氢钾1.8 mM

PBS-T
温热含有1x PBS的猎鹰管,并用切好的移液器吸头将Triton X-100(100%)加入至终浓度为0.2%。

飞食品成分
成分量/升           
玉米粉80克           
糖40克           
葡萄糖20克           
酵母提取物15克           
琼脂10克           
4-羟基苯甲酸甲酯1 g           
丙酸4毫升           
正磷酸0.6毫升           
乙醇绝对10毫升           
分别称量玉米粉,糖,葡萄糖,酵母提取物,琼脂,然后加到烧瓶中。
注意:请遵循上表中提到的不同组件的比例。

在121°C下高压灭菌20分钟,使其冷却一段时间,直到温度达到〜50-55°C。
同时,称量1克-4-羟基苯甲酸甲酯并溶于10毫升乙醇。
一旦培养基冷却至〜50-55°C(用温度计检查),就向培养基中加入4-羟基苯甲酸甲酯溶液,丙酸和正磷酸。
充分混合,然后将8-10毫升倒入每个玻璃小瓶中。
注意:避免混入气泡。不要让其冷却至〜50°C以下;否则,琼脂将开始凝固。

介质在小瓶中固化后,将紧密的棉塞放在每个小瓶上,并根据需要使用。
注意:将小瓶放在笼中进行冷却。笼子有助于避免室内杂散蝇污染小瓶。

RNA加载染料
50 µl甲酰胺

35 µl 6×DNA加载染料

15 µl超纯水(补足最终体积)

经DEPC处理的水
1毫升DEPC添加到1 ,000毫升超纯水(0.1%)的。
充分混合,盖好瓶子,在室温下放置过夜。
高压灭菌两次,使其冷却至室温。
制作等分试样并将其储存在-20°C以便使用。
TAE缓冲液(50×)
将242克Tris碱加到700毫升双蒸馏水中。
小心添加57.1毫升冰醋酸。
混合100 ml pH值为8.0的0.5 M EDTA。
调整体积至1 ,000毫升。
注意:pH值应在8.5左右,无需调整。

使用20 ml的50×TAE储备液制成1升的1×TAE缓冲液。
1×TAE缓冲液的终浓度如下:40 mM Tris碱,20 mM乙酸和1 mM EDTA
4%多聚甲醛
将50 ml 1×PBS放入干净的玻璃瓶中。
加入4 g多聚甲醛,同时将瓶子放在〜60°C的加热板上
注意:由于多聚甲醛有危险,因此请确保在通风橱内工作。

缓慢滴加1 N NaOH以溶解所有多聚甲醛。
当所有低聚甲醛都溶解在PBS中时,使用1 N HCl将pH调节至7.2-7.4。
使用1x PBS将最终体积调整为100 ml。
过滤并以10 ml等分试样的形式在0-4°C下储存低聚甲醛。
注意:多聚甲醛溶液请勿存放超过一个月。


致谢


该协议最初发表在Mehta等人的研究手稿中。(2020),并得到了科学与工程研究委员会(SERB,EMR / 2016/001819)的支持。感谢Jyotsna Kawadkar女士对果蝇中UAS-GAL4系统的准确描述。我们还要感谢IISER Bhopal的Fly设施IISER Bhopal,以及IISER的DST-FIST活细胞成像和SEM设施。


利益争夺


没有竞争利益可宣布。


伦理


研究中未使用人类受试者或小鼠模型。
对于果蝇的饲养,维护和试验,要遵循宿主研究所的动物道德规范的适用部分。

参考


布兰德,AH和佩里蒙,N。(1993)。靶向基因表达是改变细胞命运和产生显性表型的一种手段。发展118(2):401-415。
              Mehta,SJK,Kumar,V.和Mishra,RK(2020)。果蝇ELYS调节发育过程中的背动态。生物化学杂志295(8):2421至2437年。
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,LA,Hannon,GJ和Perrimon,N。(2011)。果蝇转基因RNAi的基因组规模shRNA资源。Nat Methods 8(5):405-407。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mehta, S. J., Joshi, P. A. and Mishra, R. K. (2021). Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila. Bio-protocol 11(4): e3924. DOI: 10.21769/BioProtoc.3924.
  2. Mehta, S. J. K., Kumar, V. and Mishra, R. K. (2020). Drosophila ELYS regulates Dorsal dynamics during development. J Biol Chem 295(8): 2421-2437.
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