Construction of short hairpins in Rheoswitch expression vector pNEBRX1
pNEBRX1-miR-122: A 385 bp miR-122 genomic fragment was generated by PCR using 2× Taq mix (NEB) and human genomic DNA (Novagen) using the following primers:
5' GTCACTAAGCTTCAGCTCTTCCCATTGCTCAAGATGC 3' and
5' GTCACTGGATCCGTGAGAGGCAGGGTTCAGCTAACCA 3'.
Vector pNEBR-X1(puro) was obtained by cloning a puromycin resistance cassette (PvuII-BamHI fragment) from pPur (BD Biosciences) into pNEBR-X1 (NEB). The miR-122 genomic PCR product and vector pNEBR-X1(puro) were digested with HindIII and BamHI and ligated together.
pNEBRX1-FLuc-Sh, pNEBRX1-ShM, pNEBRX1-miR-122sh and pNEBRX-Sh-1 were constructed by first annealing complementary oligonucleotides. Top and bottom oligonucleotides (50 pmoles each) were annealed by heating to 95°C and cooling slowly to 25°C in 10 mM TRIS pH 8.0. The resulting double-stranded DNA fragments with cohesive ends were ligated to appropriately digested pNEBR-X1, and transformed into competent E. coli strain NEB10beta (NEB). The following oligonucleotides were used:
FLuc-Sh BamHI top:
FLuc-Sh XhoI bottom: TCGAGGCCTAGCAGTAGCTATTTAAGTACTCAGCGTAAGTGAATAGTTTAGA
FLuc-ShM BamHI top: GATCCCCTTAGCAGAGCTGTCGAAGTACTCAGCGTAAGTGATGTCTAAACTATTCACTTACGCTAAGTACTTAAATAGCTACTGCTAGGCC
FLuc-ShM XhoI bottom:
miR-122Sh BamHI top: GATCCCCTTAGCAGAGCTGTGGAGTGTGACAATGGTGTTTGTGTCTAAACTATCAAACGCCATTATCACACTAAATAGCTACTGCTAGGCC
miR-122Sh XhoI bottom:
pNEBRX-Sh-1 HindIII top:
pNEBRX-Sh-1 BamHI bottom:
USER cloning of- pNEBRX-Sh-2, pNEBRX-Sh-3, FLuc-ShG, FLucShMG
The precise substitution of, Sh-2 Sh-3 and FLuc short hairpins for the miR-122 short hairpin, without changing the surrounding genomic DNA was accomplished using USER technology (NEB) [29
]. The plasmid vector is derived from pNEBRX1-miR-122 and contains all the sequences of the original plasmid except the miR-122 short hairpin and was generated by whole plasmid inverse PCR with the following primers containing USER sites (underlined):
5' AAACTCTGUAGCCACGAAGGTGTTAACTTCACCT 3' and
5' AATCCUTCCCUCGATAAATGTCTTGGCATCGTTTGC 3'.
The short hairpin inserts were constructed by annealing and extending oligonucleotides (listed below) with USER sites corresponding to the vector at their 5' ends (underlined), and short regions of complementarity at their 3' ends, .50 pmoles of top oligo was annealed with 50 pmoles of bottom oligo in 10 mM Tris pH7.2, by heating to 95°C for 5 minutes, then cooling slowly to 25°C. Oligonucleotides were extended using Pfu Turbo Pol Cx (Stratagene).
Vector and insert were mixed, digested with USER enzyme for 15 minutes at 37°C, annealed for 15 minutes at 25°C, then transformed into competent E. coli strain NEB5alpha. (NEB).
Oligonucleotide sequences for USER cloning:
Fluc-ShG top: ACAGAGTTUCCTTAGCAGAGCTGTCGAAGTACTCAGCGTAAGTGATGTCTAAACTAT
FLuc-ShG bottom: AGGGAAGGATUGCCTAGCAGTAGCTATTTAAGTACTCAGCGTAAGTGAATAGTTTAGAC
Fluc-ShMG top: ACAGAGTTUCCTTAGCAGAGCTGTCGAAGTACTCAGCGTAAGTGATGTCTAAACTAT
Fluc-ShMG bottom: AGGGAAGGATUGCCTAGCAGTAGCTATTTAAGTACTTAGCGTAAGT GAATAGTTTAGAC
Cloning glycogen synthase (GYS) 3' UTR
GYS 3'UTR was cloned by from HEK293-A7 cell polyA+ RNA by RT-PCR using the Protoscript First Strand cDNA Synthesis kit (NEB) and 2× Taq mix (NEB). The GYS PCR primers contained USER enzyme (NEB) cleavage sites. RT-PCR products were cleaved with USER enzyme (NEB), mixed with pNEB206A USER vector and transformed into competent E. coli strain NEB5alpha (NEB). The resulting plasmid was digested with NotI and XhoI and the 3'UTR-containing fragment was ligated into the NotI and XhoI sites of pTK-GLuc. The following oligonucleotide primers were used: GGGAAGUGCGGCCGCGTCCGCCCCACCACACTCCCCGCCTGTC (2395-2422) and GGAGACAUACCGGTTCATCTCATCTCCGGACACACTCCATTCA (3528-3500). Coordinates are from human GYS sequence, accession number NM_002103.
Cloning miR122 target into reporter plasmid pTK-GLuc
Oligonucleotides encoding two direct repeats of a sequence complementary to the miR-122 guide strand were annealed by heating to 95°C and cooling slowly to 25°C in 10 mM TRIS pH7.2. The resulting double stranded DNA fragment containing NotI and XhoI cohesive ends was ligated to pTK-GLuc (NEB) digested with NotI and XhoI to produce pTK-GLuc-miR122. Oligonucleotide sequences:
GCGGCCGCACAAACACCATTGTCACACTCCAAATCACACAAACACCATTGTCACACTCCAC and TCGAGTGGAGTGTGACAATGGTGTTTGTGTGATTTGGAGTGTGACAATGGTGTTTGTGC
All restriction endonucleases were obtained from New England BioLabs (NEB).
NIH3T3-47, HEK-293-A7 Rheoswitch cells (NEB), and NIH3T3-47/miR122 cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS), 1× non-essential amino acids, 2 mM L-glutamine, and 800 μg/mL geneticin (G418) (all from GIBCO). In addition, NIH3T3-47/miR122 cells were cultured with 1 μg/ml puromycin (Sigma). Cells were grown at 37°C, in 5% CO2 atmosphere.
NIH3T3-47/X1-miR122 (puro) stable cell lines
NIH3T3-47 Rheoswitch cells were plated in DMEM with 10% FBS (as described) in 100 mm plates. Cells were transfected at approximately 50% confluence with 15 μg pNEBRX1-miR122 (puro) per plate. 24 hours post-transfection, cells were treated with 1 μg/mL puromycin (Sigma). Cell culture medium was changed as necessary until colonies formed. Colonies were expanded and tested for RSL1-inducible expression of miR-122 by northern blot hybridization. The stable cell lines were cultured as described above with the addition of 1 μg/mL puromycin (Sigma).
Transfection and induction
For miR-122 target knockdown experiments, NIH3T3-47/miR122 cells were plated as described in 12 well plates and transfected at 50-70% confluence with 800 ng/well reporter plasmid and 100 ng/well pCMV-lacZ as a control for transfection efficiency, using Transpass D2 reagent (NEB) according to manufacturer's instructions. Reporters used were pTK-GLuc, pTK-GLuc-miR122, pTK-GLuc-GYS.
For FLuc knockdown experiments, NIH3T3-47 Rheoswitch cells (NEB) were plated as above in 24 well plates and transfected with 200 ng/well pGL3-FLuc, 100 ng/well pCMV-lacZ as transfection efficiency control and 100 ng/well of the plasmids encoding the firefly luciferase short hairpins (pNEBRX1-FLuc-Sh and -Fluc-Sh-M and pNEBRX1-FLuc-ShG and FLuc-Sh-MG).
Short hairpin expression was induced by addition of RSL1 (Intrexon) RSL1 is [(N-(2-ethyl-3-methoxybenzolyl)-N'-(3,5-dimethylbenzoytert-butylhydrazine] and has been also known as GS-E or RG-102240 [16
] or GenoStat (Millipore). A 5 mM stock solution in DMSO was diluted to a final concentration of 500 nM in the culture medium, unless otherwise noted. Controls received an equivalent volume of DMSO. DMSO final concentration was 0.1% or less. Cells were induced at 3-16 hours post-transfection and cell culture supernatants were collected for assays at 48 hours post transfection unless otherwise indicated.
Repeated Induction protocol (Figure ). NIH3T3-47/miR-122 cells were plated in 12 well plates in complete medium supplemented with 500 nM RSL1 dissolved in DMSO, or an equivalent volume of DMSO (control medium). RNA was prepared from RSL1 and DMSO treated cells after 1, 4 and 7 days in culture. For ligand withdrawal treatment, cells were cultured in complete medium containing 500 nM RSL1 for 1 day, after which it was replaced with control medium. RNA was prepared from these cells on days 4 and 7. For re-induction treatment, cells that had undergone the withdrawal treatment were cultured in control medium until day 4, after which it was replaced with medium containing 500 nM RSL1. RNA was prepared from these cells on day 7.
Total RNA was prepared from cells using TRIZOL reagent (Invitrogen) according to manufacturer's instructions.
PAGE: 10-30 μg total RNA or 60 ng microRNA marker (NEB) in 4 M urea loading buffer was heated to 95°C for 5 minutes, then loaded on 12% polyacrylamide gels (SequaGel; National Diagnostics) pre-run in 1× TBE at 250 V for 1 hour prior to loading. Gels were stained with SYBRGold (Invitrogen) to visualize RNA, electroblotted to GeneScreen Plus (Perkin-Elmer Life Sciences) at 300 mA for 30 minutes and UV crosslinked on optimum setting (Spectorlinker XL1000, Spectronics).
Oligonucleotide probes were labeled as follows: 0.5 pmol of probe oligo and 12.5 pmol of template oligo were mixed and heated to 95°C for 1 minute, incubated at room temperature for 2 minutes than placed on ice. 1 μl 10× NEBuffer 2, 5 U Klenow (exo-) (NEB), 3 μl alpha-32P-dATP (6000 Ci/mmole)(DuPont/NEN) and dH2O to 10 μl were added and incubated at 25°C for 1.5 h. Labeled oligo probe was purified over G-25 spin column (GE Healthcare) and heated to 95°C for 5 minutes before adding to hybridization. The following oligonucleotides were used:
Probe complementary to miR-122 guide strand: ACAAACACCATTGTCACACTCCA
miR-122 guide strand template: TTTTTTTTTTTGGAGTGTG
Probe complementary to miR-122 passenger strand: TGGAGTGTGACAATGGTGTTTGT
miR-122 passenger strand template: TTTTTTTTTTACAAACA
Probe complementary to Sh-1, Sh-2 and Sh-3 guide strand:GTCTGTGACTTGCACGTAC
Sh-1, Sh-2, Sh-3 guide strand template:TTTTTTTTTTGTACGTG
Probe complementary to FLuc guide strand: ATCACTTACGCTGAGTACTTCGA
FLuc guide strand template: TTTTTTTTTTTCGAAGT
Complementary regions of probe and template oligos are underlined.
U6 oligo probe: 5'CGTTCCAATTTTAGTATATGTGCTGCCGAAGCGA
] synthesized with a biotin at each end and detected using Phototope Star Detection Kit for Nucleic Acids (NEB) according to manufacturer's instructions.
Hybridization and detection
PAGE Northern blots were hybridized in UltraHyb Oligo (Ambion) at 37°C (U6) or 42°C (miR-122, FLuc) washed in 1% SDS, 50 mM sodium phosphate buffer pH 7.2 at 37°C or 42°C. Autoradiography was performed on Amersham Hyperfilm MP (GE Healthcare).
Cells were washed once in 1× PBS, followed by lysis in 1× Luciferase Cell Lysis buffer (NEB) for 15-30 minutes at 25°C with gentle agitation. Lysates were transferred to microcentrifuge tubes, cell debris was pelleted by centrifugation for 5 minutes at 4°C and lysates were stored at -20°C. 20 μL of lysate was mixed with 10 μL of 3× SDS-PAGE gel loading buffer (NEB), samples were heated to 95°C for 5 minutes and loaded on 10-20% polyacrylamide/Tris-glycine gel (Novex), run at 150 V in 1× Laemmli buffer, electroblotted to Immobilon or Immobilon-FL PVDF membrane in 1× Towbin buffer.
Tubulin control: Goat anti-alpha/beta tubulin (Cell Signaling Technologies) 1:1000 in Tris-buffered saline with 0.15% Tween20 (TBST), 2.5% milk, 2.5% BSA, followed by anti-rabbit-HRP (Cell Signaling Technologies) 1:2000. Detection used the Phototope Western detection kit (Cell Signaling Technologies);
Rabbit anti-beta tubulin (Cell Signaling Technologies) diluted 1:1000 in Odyssey buffer (LiCor) with 0.1% Tween20, followed by goat anti-rabbit-IR800 (LiCor) 1:15,000 in Odyssey buffer with 0.1%Tween 20 and 0.01% SDS. Aldolase A: Goat anti-Aldolase A (Santa Cruz Biotechnology) 1:200 in Odyssey buffer with 0.1% Tween 20 followed by donkey anti-goat-IR800, 1:15,000 in Odyssey buffer with 0.1% Tween 20 and 0.01% SDS. Fluorescent antibodies were detected using LiCor Odyssey
All assays were done in black 96 well microtiter plates and were read using either an L-max II (Molecular Devices) or a Mithras (Berthold) luminometer. Gaussia luciferase assay: 20 μL cell culture supernatant was diluted with 50 μL 1× PBS, 50 μL 1× Gaussia Luciferase Assay reagent (NEB) was injected and a 5 second integration followed a 2 second delay. Firefly luciferase: cells were washed in 1× PBS and lysed in 1× Luciferase Cell Lysis Buffer (NEB) for 15-30 minutes at 25°C with gentle agitation. 20 μL lysate was assayed with 100 μL Luciferase Assay Reagent II (Promega Dual Luciferase Assay kit) using a 10 second integration following a 2 second delay. B-gal: cells were washed in 1× PBS and lysed in 1× Luciferase Cell Lysis Buffer (NEB) or Galacto-Light Lysis buffer (Applied Biosystems). B-gal activity was assayed using the Galacto-Light kit (Applied Biosystems) according to the manufacturer's instructions. P values were calculated using a two-tailed paired t-test.