Macrophage Biology Lab San Francisco

1. Macrophage Biology LabSan Francisco RNAi II Tamara Roach Bob Rebres Bill Seaman

2. FXM: Interruption of cell signaling using RNAi • Focus: pathways proximal to Ca2+ and PIP3 • Ligands: C5a, UDP and IgG2a • Outputs: [Ca2+]i flux • changes in phosphoproteins • Approach: shRNA-based RNAi

3. Test the use of shRNA-based RNAi as a means of selective target knock-down Use Ca2+ as a readout for interruption of signaling UDP IgG2a C5a P2Y6 P2Y2 FcgRIa C5aR Fcg Gaq /Ga11 Gai2 / Gai3 ITAM ITAM PTKs Gb + Gg PLCs PI 3-kinases PIP3 Ca2+ (phosphoproteins)

4. Intracellular Ca2+ flux was used as a readout of signaling interruption [Ca2+]i flux assays on populations: adherent cells in 96-well plates 2.5 min timecourse after ligand addition Peak Response Ligand

5. C5a, UDP and IgG2a stimulate distinct patterns of intracellular Ca2+ flux UDP C5a IgG2a+ anti-IgG Ligand

6. Signaling interruption by chemical inhibitors confirmed the use of expected transduction pathways in RAW264.7 wortmannin LY249002 PP2 piceatannol PTx thapsigargin U73122 UDP IgG2a C5a EGTA P2Y6 P2Y2 FcgRIa C5aR Fcg Gaq /Ga11 Gai2 / Gai3 ITAM ITAM PTKs Gb+ Gg PLCs PI 3-kinases Ca2+ PIP3

7. Advantages of using shRNA-mediated RNAi • shRNA sequences can be stably expressed • Transduced cells can be selected by using antibiotics or • by sorting, resulting in populations of >95% carrying • vector sequences (also a potential disadvantage) • Lines can be expanded for extensive experimentation • Lines can be frozen and thawed • Replicate lines can be used to verify phenotypes • Multiple knock downs can be achieved in the same line Disadvantages……

8. To determine response phenotypes experimental vector control lines are used • Each shRNA transduction is compared to a • simultaneous control (made with empty vector). • Responses are expressed as a fraction of control • values.

9. Production of lentiviruses carrying shRNAs Vector plasmid (carries shRNA, markers, selection) VSVG-envelope plasmid 293T Gag/pol/rev plasmid Replication-deficient lentivirus carrying target shRNA sequences Infect RAW264.7 Select/sort transduced cells (GFP, hCD4, FLAG-CD7)

10. Current production is 4 experimental shRNA-transduced RAW264.7 lines per week Week 1 Week 1 2 2 3 3 4 4 5 5 Replicate assays Freeze lines Virus production Week 1 2 3 4 5 • Each of four different shRNAs is simultaneously produced and tested together with a vector control • Initiation of 4 lines every week (+control) allows the production and assay of ~16 shRNA transduced lines per month.

11. Ligand receptor knock-downs were used for initial validation of RNAi UDP IgG2a C5a P2Y6 P2Y2 X FcgRIa C5aR Fcg ITAM ITAM

12. shRNA against C5aR reduced the peak [Ca2+]i response to C5a control C5aR Knockdown of C5aR mRNA average~70% Responses to FXM ligands Line Ligand Average of 9 assays (both lines) shRNA – early algorithm, lowest KD achieved

13. shRNA against C5aR reduced [Ca2+]i flux in two independent lines control C5aR HBSS 100 nM C5a Line #1 Line #2

14. shRNA against C5aR produced a stable reduced [Ca2+]i response phenotype to C5a Longevity After freeze-thaw [Ca2+]i (nM)

15. FcgRIa receptor knock-down for validation of RNAi UDP IgG2a C5a X P2Y6 P2Y2 FcgRIa C5aR Fcg ITAM ITAM

16. shRNA against FcgRIareduced the peak [Ca2+]i response to IgG2a Knock-down of FcgRIa Responses to FXM ligands isotype control vector FcgRIa-shRNA Response (Fraction of control) C5a UDP IgG2a Log. fluorescence intensity Average of 3 assays (1 line) Median FL ratio: Knock-down/control = 0.22

17. Validation of the purinergic response requires shRNAs against 2 receptors UTP UDP X P2Y6 P2Y2 affinity: UTP>UDP affinity: UDP>UTP

18. Summary of receptor knock-downs • Target receptor expression and response can be reduced • by shRNA-mediated RNAi • Target knock-downs were stable up to 48 days • Receptor knock-downs and phenotype were reproduced • in replicate lines • Phenotypes were maintained in target lines following • freeze/thaw.