Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells
© Patel et al; licensee BioMed Central Ltd. 2012
Received: 21 October 2011
Accepted: 16 January 2012
Published: 16 January 2012
Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert.
Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358.
Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.
Cloning vectors capable of being packaged into retroviral particles for transduction into mammalian cells provide efficient tools for stably altering the genome of dividing cells. In this regard, the Moloney murine leukemia virus (MMLV)-based pBABE vector system  has been widely utilized for highly efficient stable gene overexpression in a variety of different mammalian cells with negligible off-target cellular effects. The pBABE vector consists of a bacterial origin of replication, viral elements for gene packaging, transcription and processing, and a unique restriction enzyme sequence-based cloning site http://www.addgene.org/1767/. Additionally it contains an ampicillin-resistance gene for selection in bacteria, and either puromycin, hygromycin or neomycin (G418)- resistance genes in order to select for stably transduced cell lines . This is the first retroviral transduction system that does not require helper viruses. Instead either amphotropic or ecotropic viral env gene-expressing and gag-pol gene-expressing plasmids are co-transfected along with the desired pBABE construct into an appropriate packaging cell line to produce high-titer, replication-incompetent viruses for the transfer and expression of exogenous genes in mammalian cells . The ability to use pBABE constructs as part of a three-plasmid packaging system greatly reduces the probability of recombination events leading to horizontal transfer and live virus production in the transduced lines, making it a safe and effective retroviral gene transduction system.
A major disadvantage of the pBABE plasmid is its relatively limited cloning site http://www.addgene.org/1767/. To increase cloning efficiency, two different enzymes need to be selected from the cloning site so as to generate non-complementary overhangs in the digested DNA, thus preventing self-ligation. When such a double digestion protocol is used, a series of conditions must be taken into consideration. Besides the absence of each enzyme site in the target DNA, the two enzymes should share a common set of optimal reaction conditions, including salt concentrations, digestion times and activation/inactivation temperatures. Lack of such compatible digestion conditions necessitates tedious and time-consuming sequential digestions as well as loss of DNA product during intermediate purifications. The enzymes that best fit these criteria in the pBABE multiple cloning site are SalI, BamHI and EcoRI. In our experience [2, 3], using SalI does not uniformly yield optimal digestion in combination with either BamHI or EcoRI, leading to reduced cloning efficiency. Additionally if any of these three sites, particularly BamHI or EcoRI, are internally present within the target cDNA, then the cloning process becomes extremely complicated.
Ligation-independent cloning (LIC) technology was developed to remove the conventional requirements of compatible restriction enzyme sites and exogenous enzymatic ligation during the cloning process . This methodology has been used for PCR-based amplification of genomic DNA sequences , to generate vectors for bacterial protein overexpression , for high-throughput cloning of biocatalysts from prokaryotic genomes , for the addition of variable-length C-terminal histidine tags , and for gene silencing in plant cells . However to our knowledge, there are no LIC retroviral vectors available for stably overexpressing proteins in mammalian cells.
Therefore, to increase cloning applicability and efficiency, we adapted the pBABE retroviral plasmid backbone into a LIC version denoted as pBLIC (Additional File 1, Figure S1). Unlike pBABE, cloning into this vector no longer requires conventional restriction site-generated sticky ends but instead relies on a random 12/13-sequence comprised of 3 bases. The 12/13-mer sequences are designed such that overhangs are generated using the 3' exonuclease activity of T4 polymerase. The complementary overhang sequence can be added to target cDNA inserts via PCR extension from a primer template. Because the overhang is generated by exonuclease rather than endonuclease activity and requires a free 3' end, it will not be processed even if it does happen to be internally present in the target cDNA.
Here we outline the strategy we followed to modify the pBABE plasmid vector into pBLIC. We demonstrate successful cloning of three different cDNA for proteins of different molecular weights, Bax (MW: 26 kDa), catalase (MW: 60 kDa) and p53 (MW: 53 kDa) into the adapted vector, and protein overexpression from the transduced constructs in the LNCaP human prostate cancer cell line. Thus our results support wide applicability of the modified pBLIC plasmid for mammalian retroviral transduction and protein overexpression without consideration of restriction enzymes sites or prior ligation during the cloning process. The rationale behind our basic protocol can be readily used to adapt other retroviral plasmids. Thus our method provides an easy and rapid universal method of introducing any DNA sequence of interest into the modified LIC retroviral vector, enhancing the efficiency of laboratory-scale cloning. Further the LIC retroviral system can greatly facilitate high-throughput cloning processes such as those used for the creation and verification of retroviral gene overexpression libraries where screening individual inserts for internal restriction enzyme sites is impractical.
Results and Discussion
For the sequence overhangs generated upstream of the PmlI digestion site (Figure 1), we used a random combination of A, T and C such that it was unlikely to correspond to a restriction enzyme site or coding DNA. The overhang was maintained by leaving out dCTP, dATP and dTTP from the reaction mixture, thus inhibiting T4 5' DNA polymerase activity from filling in the overhang. Conversely, digestion past the desired overhang sequence (Figure 1) was prevented by addition of dGTP to the reaction mixture, allowing polymerase activity to proceed as soon as the bold italicized G in the adaptor sequence is encountered (Figure 1). The choice of which three bases to be used to generate the random overhang is dependent on the three bases 5' of digestion site in the restriction sequence of the single cutter enzyme. For instance the restriction sequence for PmlI is CAC-GTG, where the hyphen denotes the digestion site that leads to the blunt 3' end. Thus C and A were necessarily included in the pBLIC adaptor sequence (Figure 1).
Finally we added SalI and BamHI overhang sequences to the ends of the adaptor module (Figure 1). Note that only the overhang sequences that are generated via restriction digestion (as opposed to the full palindromic consensus sequence) were added. The pBABE plasmid was digested with these two enzymes in order to remove its original cloning site and to permit ligation of the adaptor insert into the plasmid to generate pBLIC. This process restored the full BamHI site but the palindromic nature of the SalI site was destroyed (Figure 1).
Conversion of the pBABE plasmid into pBLIC was also confirmed by digestion with PmlI. Subsequent to PmlI digestion, as expected in the pBABE plasmid, the gel exhibited bands corresponding to the fully intact, nicked and linearized forms of the plasmids (Figure 2A). In contrast, the vector with the ligated LIC insert showed a single band corresponding to a linearized ~5 kb plasmid (Figure 2A). Ligation of the LIC module into the digested pBABE plasmid destroys the SalI unique cloning site present in the original plasmid. Accordingly, when both pBABE and pBLIC were digested with SalI, pBLIC no longer yielded the linearized band (Figure 2B). Finally insertion of the LIC adaptor was verified by sequencing with the SV40 promoter reverse primer 5' GAAATTTGTGATGCTATTGC 3'.
PCR primers for cloning into pBLIC
Fwd: 5' CACACCATCTCAC G GCCACCATGGACGGGTCCGGGGAGCAG 3'
Rev: 5' CTCACATTCCAC GTCAGCCCATCTTCTTCCAGATG 3'
Fwd: 5' CACACCATCTCAC G GCCACCATGGCTGACAGCCGGG 3'
Rev: 5' CTCACATTCCAC GGGTGGCTCACAGATTTGCCTTCTC 3'
Fwd: 5' CACACCATCTCAC G GCCACCATGGAGGAGCCGCAGTCAGATCC 3'
Rev: 5' CTCACATTCCAC GTCAGTCTGAGTCAGGCCCTTCTGT 3'
In this study, we demonstrate successful conversion of the pBABE retroviral transduction vector into an LIC version, pBLIC. The pBLIC vector retains the highly efficient transduction and overexpression capability of the original pBABE with the added advantages of ligation-independent cloning. Although we present here the results demonstrating gene overexpression using the converted pBABE plasmid with neomycin (G418) resistance, we have also converted the pBABE-puromycin plasmid by the same process (not shown). Furthermore the general strategy outlined above can be easily utilized to convert other retroviral plasmids such as pWZL, pLXSN or pMIG into LIC versions. Given the popularity and efficiency of the pBABE vector system for retroviral transduction and overexpression of cDNAs, we believe that the pBLIC version will simplify and extend its applications, for instance by making it a feasible vector for high-throughput cloning applications such as generation of cDNA libraries.
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' GATCC GCACACCATCTCAC GTG GAATGTGAGC 3'
3' GCGTGTGGTAGA GT GCACCTTACACTCG AGCT 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, italicizedGs 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
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, 12] 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.
RT-PCR primers for detecting cDNA transcript
Bax forward primer
5' CCCGAGAGGTCTTTTTCCGAG 3'
Bax reverse primer
5' CCAGCCCATGATGGTTCTGAT 3'
Catalase forward primer
5' CGCAGAAAGCTGATGTCCTGA 3'
Catalase reverse primer
5' TCATGTGTGACCTCAAAGTAGC 3'
p53 forward primer
5' GAGGTTGGCTCTGACTGTACC 3'
p53 reverse primer
5' TCCGTCCCAGTAGATTACCAC 3'
GAPDH forward primer
5' GACCCCTTCATTGACCTCAAC 3'
GAPDH reverse primer
5' CTTCTCCATGGTGGTGAAGA 3'
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.
- LIC :
- MMLV :
Moloney murine leukemia virus
- RT-PCR :
reverse transcriptase polymerase chain reaction.
We wish to thank Kathy Slosek and the University of Miami/Sylvester Comprehensive Cancer Center Oncogenomics Facility for DNA sequencing, and the members of the Rai laboratory for helpful discussions and general technical assistance. This study was supported in part by a James and Esther King Florida Biomed New Investigator Research grant (09KN-11) and a University of Miami Stanley J. Glaser Foundation award to P.R.
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