RNAi Libraries: shRNA


The Vienna Drosophila Resource Center has created a small number of UAS-RNAi lines using short hairpin microRNA (shRNA) technology to extend and complement the current VDRC RNAi collection. 372 shRNA lines are available as of Jan 2017 and more are in our production pipeline.


Rationale

For commonly studied genes, where there is only 1 RNAi line in the VDRC GD or KK collection at present, we aim to add a further functional RNAi line to facilitate verification of phenotypes. We chose to use the short hairpin RNAi technology as it is a simpler and more cost-effective method of creating lines than by using long double-stranded RNA. Short hairpins RNAs (shRNAs), containing a 21bp targeting sequence embedded into a micro-RNA (miR-1) backbone, have been shown to be very effective for gene knockdown in both the germline and somatic tissues (Ni et al., 2011). This technology has been used extensively by the Transgenic RNAi Project (TRiP).

To avoid direct duplication of community resources, the VDRC has collaborated with the TRiP team during shRNA design to ensure that the new VDRC lines are as distinct as possible from the TRiP resource. We have used the WALIUM20 vector (for triggering RNAi in soma and germline) in combination with the attP40 landing site, meaning that both the chromosome and insertion site differ from the majority of TRiP lines, but the insertion chromosome (2nd) and selectable marker (white) are the same as the VDRC KK RNAi collection. In addition, the hairpin sequences we have selected target a different region of the mRNA sequence and are independent of all current resources. The new lines therefore extend the range of RNAi lines available to the Drosophila research community and are complementary to the RNAi resources currently available from the VDRC, Bloomington Drosophila Stock Center (BDSC), National Institute of Genetics (NIG) and TsingHua Fly Center (THFC).

Production pipeline overview

  1. Design possible 21mers, map SNPs and select best shRNA sequence.
  2. Anneal top and bottom oligos; ligate into pWALIUM20 vector.
  3. Confirm sequence of individual constructs.
  4. Combine 50-100 constructs and inject as a pool into w- embryos for targeted phiC31-mediated integration.
  5. Select w+ transformants and balance transgenic line.
  6. Sequence transgenic line to confirm identity of shRNA.
  7. Make line available via VDRC online ordering system.
VDRC shRNA lines
Vector pWALIUM20 (Perkins et al., 2015)
map and sequence
Landing site attP40 (Markstein et al., 2008)
Integration locus 2nd chromosome @ 25C6
Selectable marker white
Knock-down in soma and germline

Hairpin design

  • 21nt siRNA oligos were designed to target exons common to all isoforms of a gene using the DSIR website tool (http://biodev.extra.cea.fr/DSIR/DSIR.html) and an in-house off-target analysis script (Thomas Burkard, IMP/IMBA, Vienna).
  • SNPs were called with GATK tool (Broad Inst.) using whole genome resequencing data from 10 Drosophila samples, aligned against the UCSC dm3 genome assembly.
  • Overlap of a shRNA with a SNP was detected with an in-house script (BioComp, Vienna Biocenter Core Facilities).
  • For each gene, the best shRNA sequence was selected based on a combination of highest predicted activity, lowest off-target probability and absence of common SNPs in a critical position.
  • A small number (5%) of shRNAs exactly matching those already created or planned by TRiP (pers. comm. L.Perkins, C. Yu) were eliminated to avoid duplication of resources.

Construct generation

71 base oligos were designed according to the methods used by the TRiP team by concatenating the following:

Top strand oligo = 'ctagcagt', the sense-strand oligo (21 base pairs), 'tagttatattcaagcata', the anti-sense oligo, and 'gcg'.

Bottom strand oligo = 'aattcgc', the sense-strand oligo, 'tatgcttgaatataacta', the anti-sense oligo, and 'actg'.


Top and bottom 71mer oligos were annealed, creating overhangs for ligating into pWALIUM20 via the NheI and EcoRI sites, as described by Ni et al., 2011.

Ligation reactions were transformed into NEB10beta competent cells followed by colony PCR and sequencing to identify clones with the correct shRNA sequence.

PCR primers for confirming shRNA sequence:
fwd: 5'-ACCAGCAACCAAGTAAATCAAC-3'
rev: 5'-TAATCGTGTGTGATGCCTACC-3'

Sequencing primer:
5'-ACCAGCAACCAAGTAAATCAAC-3'

Only those constructs confirmed to have correct DNA were used for injection. Plasmid DNA was prepared from minipreps using GeneJET columns (Fisher Scientific). Typically, 50-100 constructs were pooled at equal concentrations, purified with AMPure XP beads (Agencourt) and resuspended at 200-250ng/ul.


Landing site

All fly shRNA stocks created by the VDRC are integrated into the attP40 genomic landing site on the left arm of the second chromosome at 25C6. The selectable marker is white, so the presence of the UAS-shRNA transgene can be confirmed by the stock having red eyes.

The attP40 site was chosen because it has been shown to provide high levels of induced expression of transgenes, yet maintain a low basal expression when the transgenes are not induced (Markstein et al., 2008).

The majority of the TRiP RNAi stocks have insertions in the attP2 genomic landing site on chromosome 3 with vermilion as the selectable marker, whereas the VDRC shRNA and KK lines have the RNAi construct inserted on chromosome 2 with white as the selectable marker.


Injections and crossing scheme

Since only one attB insert can integrate into an attP site via phiC31-mediated recombination (Groth et al., 2004), we injected constructs in pools of 50-100. Constructs were microinjected into embryos derived from a cross between males homozygous for the attP40 integration site (Markstein et al., 2008) and females homozygous for a fusion gene encoding the PhiC31 integrase under the control of the vasa promoter (vas-integ), which provides a maternal source of integrase (Bischof et al., 2007). Single males derived from these embryos were crossed to w- females, and males carrying the inserted construct (identified by their w+ eye colour) were selected; note that the source of integrase is also removed in this step. These w+ males were crossed to w;Sp/CyO females to establish balanced, homozygous stocks. The majority of stocks are homozygous viable, but a small number are homozygous sterile or lethal and still have CyO present (status listed under 'Viability' for each stock).

For crossing scheme figure see here.


Stock characterization and verification

In order to identify which of the pooled constructs were inserted in each transgenic line, genomic DNA was extracted as soon as w+ flies could be sacrificed, either directly after setting up the cross to the Sp/CyO balancer stock, or during the subsequent crosses to create a homozygous stock. The inserted DNA was amplified across the entire hairpin and sequenced to assign the stock to a construct.

PCR primers for confirming shRNA sequence:
fwd: 5'-ACCAGCAACCAAGTAAATCAAC-3'
rev: 5'-TAATCGTGTGTGATGCCTACC-3'

Sequencing primer:
5'-ACCAGCAACCAAGTAAATCAAC-3'

After genetic crosses to balance the stock and make it homozygous, the sequence of the transgene was again verified (using the same primers) before making the completed stock available at www.vdrc.at.


Control lines

Currently, the best control for the shRNA lines is VDRC_ID 60200, containing an empty pWALIUM20 vector (no hairpin sequence) integrated into the attP40 landing site, created in the same way and with same gene genetic background as the shRNA lines. It can be found under 'Browse RNAi Toolkit Stocks'. We are creating an shRNA-EGFP line in the same way to provide a further control. Alternatively, we suggest using a VDRC shRNA line which is known NOT to function in the process or pathway being studied.


Acknowledgements

We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing the pWALIUM20 plasmid vector used in the creation of the VDRC shRNA lines.

We are especially grateful to Liz Perkins, Claire Hu and Norbert Perrimon (TRiP, Harvard Medical School) for their helpful discussions and for coordinating the design of the shRNAs.

Please acknowledge the VDRC for providing the lines!