Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing

Abstract

As a general strategy for function-based gene identification, an shRNA library containing ≈150 shRNAs per gene was enzymatically generated from normalized (reduced-redundance) human cDNA. The library was constructed in an inducible lentiviral vector, enabling propagation of growth-inhibiting shRNAs and controlled activity measurements. RNAi activities were measured for 101 shRNA clones representing 100 human genes and for 201 shRNAs derived from a firefly luciferase gene. Structure-activity analysis of these two datasets yielded a set of structural criteria for shRNA efficacy, increasing the frequencies of active shRNAs up to 5-fold relative to random sampling. The same library was used to select shRNAs that inhibit breast carcinoma cell growth by targeting potential oncogenes. Genes targeted by the selected shRNAs were enriched for 10 pathways, 9 of which have been previously associated with various cancers, cell cycle progression, or apoptosis. One hundred nineteen genes, enriched through this selection and represented by two to six shRNAs each, were identified as potential cancer drug targets. Short interfering RNAs against 19 of 22 tested genes in this group inhibited cell growth, validating the efficiency of this strategy for high-throughput target gene identification.

Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ2 renders tumor cells dependent on its paralogous gene COPZ1

“…To enable doxycycline-inducible GSE expression, the cell lines were modified by the introduction of tTR-KRAB, a tetracycline/doxycycline-sensitive repressor. Cells transduced with the GSE library were subjected to selection for doxycycline-dependent resistance to BrdU suicide, a procedure that selects for cells carrying growth-inhibitory GSEs (5,16). The library-derived cDNA fragments were amplified by PCR from genomic DNA of the unselected and BrdU-selected cells.…”

“…These sequences include antisense cDNAs (10), genetic suppressor elements (GSEs, short cDNA fragments that express dominant negative protein fragments or antisense RNA segments) (5,11,12), and shRNAs that inhibit gene expression through RNA interference (13)(14)(15)(16). In the present article, we report the identification, through GSE selection, of a tumor-specific gene target, inhibition of which kills both proliferating and nondividing tumor cells.…”

NLRP3 inflammasome: Pathogenic role and potential therapeutic target for IgA nephropathy

“…Plasmids of shNLRP3-luciferase was generated by conjugating shNLPR3 (Santa Cruz) with luciferase as described previously55. The plasmids in 200 μl of saline was mixed with 200 μl of SonoVue® microbubbles (Bracco), and the mixture injected into mice via the tail vein, followed by application of transcutaneous ultrasound to the back at the level of the kidney using a Sonopuls 590 at 1 MHz (Ernaf-Nonius) as described previously50.…”

Transcriptomes and shRNA Suppressors in a TP53 Allele–Specific Model of Early-Onset Colon Cancer in African Americans

“…On Day 1, RKO clones were seeded at 1.5×10 6 cells in one T-150 flask per cell line, and incubated at 37°C for 24 hrs. On Day 2, each cell line was infected with a lentiviral shRNA library at MOI~0.8 as described (31) with the addition of 8.0ug/mL Polybrene for 24 hrs. The library consists of 27,500 shRNAs targeting 5,043 genes (Decipher Project – Human Module 1, Signaling Pathway Targets).…”