LNCaP cells were cultured in RPMI-1640 medium containing 5% fetal bovine serum and 100 units/ml penicillin-streptomycin. H358 cells were cultured in RPMI-1640 in 10% fetal bovine serum and 100 units/ml penicillin-streptomycin. HEK-293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 100 units/ml penicillin-streptomycin. All cell culture reagents were obtained from Gibco, Invitrogen.
Design of the LIC adaptor segment
The LIC segment was designed to contain SalI and BamHI overhangs to allow ligation into the pBABE cloning site. It also contained the following 12/13-mer sequences to create overhangs via T4 exonuclease activity (13 base overhang on the left below, 12 base overhang on the right):
5' GATCCGCACACCATCTCACGTGGAATGTGAGC 3'
3' GCGTGTGGTAGAGTGCACCTTACACTCGAGCT 5'
The bolded letters indicate the complementary SalI and BamHI sites used to ligate the segment into the pBABE backbone. The underlined site is for the unique blunt cutter PmlI to generate the ends/starting site for 3' T4 polymerase exonuclease activity. The 12/13-base sticky ends generated by this activity are italicized. The bold, italicized Gs are stopping points for T4 exonuclease activity. This is accomplished by adding only dGTP to the reaction mixture. Thus the chemical equilibrium for T4 polymerase shifts from the 3'-> 5' exonuclease activity to the 5'-> 3' polymerase activity only once the italicized bolded G is encountered by T4 polymerase as the other complementary nucleotides to the italicized overhang are absent. The above oligonucleotides (Integrated DNA Technologies) were used to generate the adaptor segment by annealing in a 1:1 ratio (5 μl of 1 μg/μl each in NEB Buffer 2).
Construction and preparation of the modified backbone vector
The pBABE-neomycin vector was digested with BamHI and SalI to remove the original cloning site and the resulting segment was gel purified using the Qiagen gel purification kit according to manufacturer instructions. The annealed LIC adaptor segment (described above) was diluted 1:10 and mixed with 50 ng of the purified digested pBABE vector prior to ligation using a Rapid DNA ligation kit (Roche) as per manufacturer's instructions. The resulting pBLIC plasmid was digested with PmlI to generate the linearized plasmid. This was subsequently treated with T4 polymerase (NEB). Treatment conditions were as follows: 0.4 pmol digested vector, 2 μl 10X Buffer 2 (NEB), 2 μl dGTP (Roche), 2 μl 100 μM dithiothreitol (DTT, Sigma), 0.4 μl T4 Polymerase (NEB). DNAse- and RNAse-free water (Gibco, Invitrogen) was used to make up a total volume of 20 μl. This solution was incubated at 22°C for 40 minutes and then at 75°C for 20 minutes to inactivate the T4 polymerase.
General primer design for cDNA of interest
In order to generate the cDNA fragment to be cloned into the pBLIC backbone, the following PCR primer design was utilized to contain complementary sequences to the T4 exonuclease activity-generated overhangs in pBLIC:
Sense: 5' CACACCATCTCACG-GCCACC-the first 20 bases of cDNA starting with ATG
Antisense: 5' CTCACATTCCACG-20 bases from the 3' end of the cDNA
The italicized nucleotides in the forward primer correspond to the Kozak sequence [11
], to improve translational efficiency. For primers specific to Bax, catalase and p53, see Table .
PCR amplification of insert and addition of complementary overhangs
Samples for PCR amplification were made by adding 50 ng of DNA, 1 μl of 10 μM dNTP, 5 μL of 10X PCR buffer, 1.5 μl of 50 mM MgCl2, 1 μl each of forward and reverse primer at a 10 μM final concentration, 0.5 μl of Platinum taq DNA polymerase (Invitrogen) and dH2O for a 50 μl final reaction volume. Gradient PCR (Eppendorf) with annealing temperatures between 55-67°C and 35 cycles was used for optimal primer extension. Unincorporated dNTPs from the PCR samples were removed using a gel purification kit (Qiagen). This was subsequently treated with T4 polymerase (NEB). Treatment conditions were as follows: 0. 2 pmol annealed DNA, 2 μl 10X Buffer 2 (NEB), 2 μl dCTP (Roche), 1 μl 100 μM DTT, 0.4 μl of T4 Polymerase (NEB) and DNAse- and RNAse-free water (Gibco, Invitrogen) to make a total volume of 20 μl. This solution was incubated at 22°C for 40 minutes and then at 75°C for 20 minutes to inactivate T4 polymerase.
Bacterial transformation and amplification of the pBLIC cDNA retroviral mammalian construct
The T4-treated pBLIC construct (0.04 pmol) and cDNA fragment (0.04 pmol) were mixed together using 1 μl of the former and 2 μl of the latter, and incubated for 5 minutes at 22°C. Instead of a ligation step, 1 μl of 25 mM EDTA was added to a final solution volume of 4 μl for a further 5-minute incubation period to stabilize the non-covalent interactions between the DNA backbone and insert. Approximately 1 μl of this mixture was used to transform recombination-deficient competent XL-10 Gold bacteria (Stratagene) and plated on ampicillin-agar plates (final ampicillin concentration: 100 μg/ml). Competent colonies were selected for inoculation in 100 μg/ml ampicillin-containing LB media, and the resulting DNA plasmid was purified using Qiagen DNA mini and midi prep kits.
Verification of cloned construct by enzymatic treatment and DNA sequencing
Purified cloned plasmids were analyzed for the appropriate cDNA insert by digesting the constructs with HindIII to detect a shift in size equal to the size of Bax, catalase or p53. Additionally, using sequencing primers against pBABE (pBABE5': CTTTATCCAGCCCTCAC / pBABE3': ACCCTAACTGACACACATTCC), constructs were sequenced at the University of Miami Oncogenomics Facility to verify that the target cDNAs were present in the respective cloned pBLIC construct (Additional File 1
, Figure S2).
Retroviral transduction of constructs
The pBLIC-Bax, pBLIC-catalase and pBLIC-p53 constructs were transduced as described previously [2
] into the LNCaP human prostate cancer cells or H358 human lung cancer cells. Briefly viral particles were produced by co-transfecting 3 μg MuLV gag-pol-expressing plasmid pUMVC, 300 ng envelope protein-expressing plasmid pCMV.VSV-G and 3-4 μg target construct into HEK 293T cells. The transfection complex was produced in serum-free DMEM media via the transfection agent Fugene®
6 (Roche) at Fugene (μl): total DNA (μg) ratio of approximately 2:1. The viral supernatants from 293T cells were harvested at 48 hours and 72 hours and applied to the LNCaP cells for 6-8 hours in the presence of 6 μg/ml protamine sulfate. At 48 hours after the last transduction, 500 μg/ml G418 was added to the cell culture media and to a mock-transduced plate of cells. Cells were subsequently continuously selected in G418-containing media to enrich for successfully transduced cells.
Total RNA was isolated using the RNAqueous-4PCR Kit (Ambion) as per manufacturer instructions. A total of 0.25 μg purified RNA was reverse-transcribed using a random primer to obtain cDNA via the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The cDNA was used as a template for PCR amplification using AmpliTaq Gold® 360 Master Mix (Applied Biosystems) via the primers listed in Table . The reactions were run on a 1.5% agarose gel and bands were visualized by ethidium bromide staining. GAPDH was used as an internal loading control.
RT-PCR primers for detecting cDNA transcript
Western Blotting of proteins
Protein lysates were made from harvested cell pellets by resuspending in sodium fluoride (NaF; 50 mM Tris PH 7.5, 150 mM NaCl, 1% Nonidet P-40, 50 mM NaF) lysis buffer (10 μL 0.1 M sodium vanadate (NaVO3), 20 μL 50× protease inhibitor, 9 μL of 100 mM phenylmethylsulfonyl fluoride (PMSF), 1 μL of 1 M DTT per 1 ml NaF base buffer). Samples were incubated on ice for 30 min and then centrifuged at 14,000 rpm for 20 min. Protein concentrations were determined using the Bradford assay (5X Bradford Reagent, Biorad). Subsequently 35 μg of protein from each sample was prepared for immunoblotting on a 4-12% Bis-Tris gradient pre-cast gel (Nupage, Invitrogen) on a Novex immunoblotting module (Invitrogen). The gel was run at 120 V for 2 hrs on ice and then was transferred to a PDVF membrane (Immobilon, Millipore) at 35 V for 1.75 hrs in cold room. After transfer, the membrane was immersed in Ponceau reagent (Sigma) to assess relative loading among the various lanes. The membrane was blocked in 5% non-fat dry milk in 0.1% Tween/1X TBS (TBST), incubated with the appropriate antibodies: Bax (1:4000, sc-493, Santa Cruz Biotechnology Inc.), catalase, (1:4000, ab16731, Abcam), p53 (1:1000, sc-126, Santa Cruz Biotechnology Inc.), and GAPDH (1:4000, ab9485, Abcam). Subsequently the blots were washed in 0.1% TBST. After the incubation period in the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Amersham), the blots were again washed in 0.1% TBST. Blots were then exposed to autoradiographic films and developed with the ECLPlus Western Chemiluminescent Detection System (GE Healthcare, Amersham) to determine levels of protein expression.