CaltechAUTHORS
  A Caltech Library Service

A Random shRNA-Encoding Library for Phenotypic Selection and Hit-Optimization

Wang, Yongping and Wang, Yun E. and Cotticelli, M. Grazia and Wilson, Robert B. (2008) A Random shRNA-Encoding Library for Phenotypic Selection and Hit-Optimization. PLoS ONE, 3 (9). e3171. ISSN 1932-6203. PMCID PMC2525836. https://resolver.caltech.edu/CaltechAUTHORS:WANplosone08

[img]
Preview
PDF - Published Version
Creative Commons Attribution.

441Kb
[img] MS Word (Table S1. (0.03 MB DOC) Enrichment for true-positive shRNA sequences after various rounds of selection and re-screening, assuming a true positive rate of only 1 in 1 million, false positive rates of either 1% (left) or 10% (right), and no heritability..) - Supplemental Material
Creative Commons Attribution.

31Kb
[img]
Preview
Image (TIFF) (Figure S1. (0.16 MB TIF) Sequencing data of the random library) - Supplemental Material
Creative Commons Attribution.

152Kb
[img]
Preview
Image (TIFF) (Figure S2. (0.08 MB TIF) Vector preparation for cloning of the shRNA-encoding cassette) - Supplemental Material
Creative Commons Attribution.

81Kb
[img]
Preview
Image (TIFF) (Figure S3. (0.04 MB TIF) 8p offers delayed protection of FL5.12 cells from IL3 withdrawal) - Supplemental Material
Creative Commons Attribution.

34Kb
[img]
Preview
Image (TIFF) (Figure S4. (0.06 MB TIF) 1p, 3p and 8p do not offer FL5.12 cells a growth advantage. Cells were infected with 1p, 3p, 8p, or pSiren and compared with cells infected with the same random control shRNA (ctrl)) - Supplemental Material
Creative Commons Attribution.

60Kb
[img]
Preview
Image (TIFF) (Figure S5. (0.02 MB TIF) Effects of the hit shRNAs are long lasting. Cells were cultured in IL3+ media continuously for 4 months. IL3 was then withdrawn for 20 hours, and protective effects were still seen (p = 0.0087 for 1p vs. ctrl, p = 0.073 for 3p v) - Supplemental Material
Creative Commons Attribution.

18Kb
[img]
Preview
Image (TIFF) (Figure S6. (0.03 MB TIF) 10 random clones offered no protection. FL5.12 cells were transduced with 10 additional random clones from the 300K library, along with pSiren, or the 300K library itself. IL3 was withdrawn for 15 hours, and the survival percent) - Supplemental Material
Creative Commons Attribution.

27Kb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:WANplosone08

Abstract

RNA interference (RNAi) is a mechanism for inhibiting gene expression through the action of small, non-coding RNAs. Most existing RNAi libraries target single genes through canonical pathways. Endogenous microRNAs (miRNAs), however, often target multiple genes and can act through non-canonical pathways, including pathways that activate gene expression. To interrogate all possible functions, we designed, synthesized, and validated the first shRNA-encoding library that is completely random at the nucleotide level. Screening in an IL3-dependent cell line, FL5.12, yielded shRNA-encoding sequences that double cell survival upon IL3 withdrawal. Using random mutagenesis and re-screening under more stringent IL3-starvation conditions, we hit-optimized one of the sequences; a specific nucleotide change and the creation of a mismatch between the two halves of the stem both contributed to the improved potency. Our library allows unbiased selection and optimization of shRNA-encoding sequences that confer phenotypes of interest, and could be used for the development of therapeutics and tools in many fields of biology.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1371/journal.pone.0003171DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525836/PubMed CentralArticle
Additional Information:© 2008 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: July 3, 2008; Accepted: August 11, 2008; Published: September 9, 2008. We thank Zissimos Mourelatos for helpful discussions. This work was funded by research grants (to R.B.W.) from the National Institute for Neurological Disorders and Stroke (R21-NS-053546) and the Friedreich's Ataxia Research Alliance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: Conceived and designed the experiments: YW RBW. Performed the experiments: YW YEW MGC. Analyzed the data: YW RBW. Wrote the paper: YW RBW. Competing interests: The content of this work is the subject of a pending patent. Therefore, publication of the results could result in potential commercial gain. The authors (Yongping Wang and Robert Wilson) are the holders of this patent. Supporting Information: Table S1. (0.03 MB DOC) Enrichment for true-positive shRNA sequences after various rounds of selection and re-screening, assuming a true positive rate of only 1 in 1 million, false positive rates of either 1% (left) or 10% (right), and no heritability of false positives. Figure S1. (0.16 MB TIF) Sequencing data of the random library. (A) Sequencing results from the shRNA library before the creation of the non-complementary loop. At this stage, PmeI digest allowed the two halves of the stem to be separated and sequenced more easily. The electropherogram confirms all features of the cassette design. (B) Sequences of 40 random clones from the library. Note the last base is a G for all sequences, as discussed in the main text. In a perfectly random scenario, therefore, there should be 14 A/Ts and 15 G/Cs. The 40 clones showed a distribution of 14.78 A/Ts and 14.22 G/C, on average. Thus there is a very slight bias towards A/T. No obvious patterns in these sequences can be discerned. Figure S2. (0.08 MB TIF) Vector preparation for cloning of the shRNA-encoding cassette. (A) The cassette was initially designed for cloning into pSuper (Oligoengine); shown here are the steps used to prepare pSuper for blunt-end/NotI cloning. A linker containing the BbsI site was cloned into pSuper cut with BglII and MluI. The linker also contained the SnaBI site for the purpose of checking the ligation since there is another SnaBI site in pSuper. The altered pSuper was then digested with BbsI, which is a downstream cutter, and then filled-in with Klenow to form blunt ends on both sides. BbsI was positioned precisely on the linker, such that after the digest and fill-in, the blunt end is at the correct distance from the promoter per the manufacturer's instructions. The blunt-blunt vector was then cut with NotI to form the blunt-end/NotI pSuper, ready for ligation of the shRNA-encoding cassette (Figure 1B) (B) Transfer of the library en bloc into pSiren (Clontech) to ensure consistent GFP expression after retroviral infection and integration. The standard restriction sites for cloning into pSiren are BamHI and EcoRI. We introduced a NotI site between the BamHI and EcoRI sites using a primer with all three sites in the correct order. This primer also mismatches the pSiren template by “jumping over” two bases highlighted in the figure. These two bases needed to be eliminated because the cassette excised from pSuper with BglII-NotI had two extra bases, “CC,” after the BglII site (see part (A)). Therefore, paired with an appropriate upstream primer, we introduced a NotI site and eliminated two bases in pSiren. The modified vector was then cut with BamHI and NotI, and was ready to accept the BglII-NotI cassette from pSuper, with the correct spacing from the PolIII promoter. Note that BamHI and BglII have compatible cohesive ends. The two primer sequences are: “RINotBam” 5′-CTTGAATTCGCGGCCGCTTGGATCCGTCCTTTCCACAAG-3′, and “SirenBgl” 5′-CCGGAATTGAAGATCTGGG-3′. Figure S3. (0.04 MB TIF) 8p offers delayed protection of FL5.12 cells from IL3 withdrawal. Initially, 8p did not offer protection (Figure 3), but its effect became apparent one week later. Survival percentages were obtained in the same manner as for all other survival figures, after IL3 starvation of 22 hours in this experiment. (p = 0.019 for 1p vs. ctrl, p<0.01 for 3p or 8p vs ctrl) Data are means +/− SD. Figure S4. (0.06 MB TIF) 1p, 3p and 8p do not offer FL5.12 cells a growth advantage. Cells were infected with 1p, 3p, 8p, or pSiren and compared with cells infected with the same random control shRNA (ctrl). GFP% was 20–25% after the infections. Cells were then seeded at 500,000/2 ml (relative cell number = 1) in a 12-well plate and allowed to grow for three days. None of the three hit shRNAs offered any growth advantage over the random control shRNA. The growth rate was also comparable to the GFP-negative cells in the same culture. Data are means +/− SD. Figure S5. (0.02 MB TIF) Effects of the hit shRNAs are long lasting. Cells were cultured in IL3+ media continuously for 4 months. IL3 was then withdrawn for 20 hours, and protective effects were still seen (p = 0.0087 for 1p vs. ctrl, p = 0.073 for 3p vs. ctrl, p = 0.012 for 8p vs. ctrl). Data are means +/− SD. Figure S6. (0.03 MB TIF) 10 random clones offered no protection. FL5.12 cells were transduced with 10 additional random clones from the 300K library, along with pSiren, or the 300K library itself. IL3 was withdrawn for 15 hours, and the survival percentage was similar in all cases. A positive control was not included in this experiment; however, we have performed such starvation assays more than 50 times under different conditions, and the survival percentages shown in this figure are seen consistently. Data are means +/− SD.
Issue or Number:9
PubMed Central ID:PMC2525836
Record Number:CaltechAUTHORS:WANplosone08
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:WANplosone08
Usage Policy:This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ID Code:11847
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:06 Oct 2008 22:44
Last Modified:03 Oct 2019 00:23

Repository Staff Only: item control page