Cells that silenced an shRNA outgrew those expressing it, masking phenotypes of essential genes; this paper linked a Tet-regulated miR30-shRNA to dsRed and a constitutive Venus marker (TRMPV) to track and sort true expressers. Rpa3 knockdown then kept 75% of treated AML mice disease-free for 22 weeks and gave over 250-fold depletion in a pooled in vivo screen.
The TRMPV retroviral vector (TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-NeoR) produces two transcripts: the TRE promoter drives a dsRed reporter together with a miR30-embedded shRNA, and the PGK promoter drives constitutive Venus and, through an IRES, the neomycin resistance gene. Cells that express the shRNA are identified as Venus+dsRed+. Variants use TRE-tight (reduced basal activity), alternative drug markers, or alternative fluorescent proteins.
02
In competitive proliferation assays with Rosa26-rtTA-M2 mouse embryonic fibroblasts on doxycycline, Rpa3 shRNAs deplete Venus+dsRed+ cells strongly but Venus+ cells only moderately, because Venus+dsRed− clones that fail to induce the TRE accumulate and escape Rpa3 knockdown. Quantifying Venus+dsRed+ cells therefore measures the antiproliferative effect of an shRNA more sensitively than the constitutive Venus marker alone.
03
Placing rtTA3 upstream of an IRES in a bicistronic transcript with the oncogene MLL-AF9 (rtTA3-IRES-MLL-AF9) selects for strong rtTA expression through the proliferative advantage of the oncogene, and gives more robust rtTA expression than the reverse orientation. Together with Luci-IRES-NrasG12D, this produces an MLL-AF9;NrasG12D acute myeloid leukemia model in which TRMPV-shRpa3 depletes dsRed+Venus+ cells on doxycycline.
04
The single-vector TRMPVIR (TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-rtTA3) delivers a TRE-driven shRNA and rtTA3 together. PGK drives basal rtTA3 and, once doxycycline activates the TRE, the TRE transcript supplies more rtTA3 through its downstream IRES, forming a positive feedback loop. In Eμ-myc;Trp53−/− lymphoma cells, TRMPVIR-shRpa3 induces dsRed on doxycycline and Venus+dsRed+ cells are outcompeted, whereas shRen cells are maintained.
05
In mice transplanted with Tet-On MLL-AF9;NrasG12D leukemia carrying TRMPV, the weak shRpa3.53 and the control shRen allow death within about 17 days on doxycycline, whereas intermediate shRpa3.276 and strong shRpa3.455 delay disease and prolong survival. All relapses are dsRed-depleted, with outgrowth of Venus+dsRed− or double-negative clones that escaped shRNA expression, and shRpa3.53 does not significantly reduce Venus+dsRed+ frequency in vivo.
06
Clonal TRMPV-shRpa3.455 MLL-AF9;NrasG12D leukemias regress rapidly when doxycycline is given at disease onset or at an advanced stage, with leukemic blasts cleared from bone marrow, spleen and liver within 4 days. All doxycycline-treated mice reach complete remission, and 75% remain healthy with no luciferase signal for 22 weeks even when doxycycline is withdrawn after 40 days; relapsed leukemias are dsRed-negative.
07
A pooled in vivo negative-selection screen of 824 TRMPV shRNAs in Tet-On MLL-AF9;NrasG12D leukemia at predominantly single retroviral integration detects over 95% of shRNAs in each untreated mouse. Sorting Venus+dsRed+ cells from doxycycline-treated mice deepens the detectable depletion of the three Rpa3 control shRNAs four- to tenfold (ratios below 0.01 rather than below 0.1), over 250-fold in total, compared with two- to tenfold typical of microarray-based screens, and all three rank among the top 25 depleted shRNAs.
08
miR30-shRNAs are cloned into TRMPV as 116-nucleotide XhoI–EcoRI fragments generated by PCR from 97-mer oligonucleotides with 5′ miR30-XhoI and 3′ miR30-EcoRI primers. Each shRNA is named for the first nucleotide of its 22-nucleotide target site in the mRNA.
1 / of8
Abstract
Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.
Choosing a selection results in a full page refresh.
