Materials and Reagents

1.5 ml MaxyClear Microtubes (Axygen, catalog number: MCT-150-C)
0.2 ml Polypropylene PCR Tube (Axygen, catalog number: PCR-0208-C)
Pipette tips (Corning, Axygen^®)
0.22 μm Millipore filters (Biosharp, BS-QT-011)
Petri dishes
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)
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.
EcoRI-HF (NEB, catalog number: R3101L)
NheI-HF (NEB, catalog number: R3131L)
SpeI (NEB, catalog number: R3133L)
XbaI (NEB, catalog number: R0145L)
Alkaline Phosphatase, Calf Intestinal (CIP, NEB, catalog number: M0290S)
AxyPrep^TM DNA Gel Extraction Kit (Corning, Axygen, catalog number: AP-GX-250)
T4 DNA ligase (NEB, catalog number: M0202L)
Trans5α Chemically Competent Cell (Transgen Biotech, catalog number: CD201-01)
Go Tag Green Master Mix, 2x (Promega, catalog number: 000179370)
AxyPrep^TM Plasmid Miniprep Kit (Corning, Axygen, catalog number: AP-MN-P-250)
PurePlasmid Mini Kit (CWBiotech, catalog number: CW0500M)
Primers sequences:
U-F: 5’-GCTGAGAGCATCAGTTGTGA-3’; Ftz: 5’-TAATCGTGTGTGATGCCTACC-3’
Buffer PB (QIAGEN, catalog number: 154051779)
LB Broth, Miller (Luria Bertani) (Becton Dickinson)
Agar (Beyotime)
Ampicillin (Solarbio, catalog number: A8180)
Tris-HCl (AMRESCO, catalog number: 1185-53-1)
EDTA (AMRESCO, catalog number: 60-00-4)
NaCl (AMRESCO, catalog number: 7647-14-5)
KCl (AMRESCO, catalog number: 7447-40-7)
Ethidium bromide (Sigma, catalog number: E8751)
50x TAE (Double Helix, catalog number: P0309A)
Sodium phosphate buffer stock solution (pH 6.8) (see Recipes)
10x Annealing Buffer (see Recipes)
Injection Buffer (see Recipes)
LB (Luria Bertani) medium (see Recipes)
LB/ampicillin plates (see Recipes)
1x TAE (see Recipes)
1.5% agarose gel (see Recipes)

Equipment

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

Software

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

Procedure

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 AxyPrep^TM 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 AxyPrep^TM 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 AxyPrep^TM 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 AxyPrep^TM 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.
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

10x Annealing Buffer (store at RT)
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
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
LB (Luria Bertani) medium
Dissolve 25 g of the powder in 1 L of ddH₂O, mix thoroughly
Autoclave at 121 °C for 15 min
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
1x TAE
20 ml 50x TAE
980 ml ddH₂O
Store at room temperature
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

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