Vector construction
The assay plasmid for detecting chromosomal deletions was generated as follows. Plasmid pBB-B304-P333 has been described [
4] and will be referred to as pBB-TP(BP). This plasmid contains the full-length TP901-1
attP and
attB recognition sites flanking the 3.5 kb
lacZ gene. The 64 bp R4
attP site was generated by kinasing and annealing the oligonucleotides 5'-CCGGGCATGTTCCCCAAAGCGATACCACTTGAAGCAGTGGTACTGCTTGTGGGTACACTCTGCGGGTGTAC-3' and 5'-CCGGGTACACCCGCAGAGTGTACCCACAAGCAGTACCACTGCTTCAAGTGGTATCGCTTTGGGGAACATGC-3', followed by ligation into the
Xma I site of pBB-TP(BP), creating the plasmid pBB-TP(BP)-R4(P). Into the
Sac II site of this plasmid, the 224 bp φC31
attP site was cloned as an
Xba I-
Sca I fragment from plasmid pBCPB+ [
1], resulting in the plasmid pBB-TP(BP)-R4(P)-φC31(P). Into the
Xho I site of this plasmid, the 313 bp φC31
attB site was cloned as an
Xho I fragment released from pBCPB+, generating plasmid pBB-TP(BP)-R4(P)-φC31(BP). Into the
Bam HI site of this plasmid, the 295 bp R4
attB site was ligated as an
Xho I fragment released from the plasmid pBC-R4PB [
3], creating the plasmid pBB-TP(BP)-R4(BP)-Phi(BP). Lastly, a 2.0 kb
Sal I-
Nru I fragment from pEF [
4], encoding the gene for hygromycin resistance driven by the TK promoter, was cloned into the
Age I site of pBB-TP(BP)-R4(BP)-φC31(BP), generating the assay plasmid pBB-TP(BP)-R4(BP-φC31(BP)-Hyg, referred to as plasmid p3BP.
Each integrase was independently cloned into the expression plasmid pIRES-hrGFP-1a (Stratagene, La Jolla, CA). The TP901-1 integrase was cloned into the
Eco RI-
Xho I sites of pIRES-hrGFP-1a as a 1.5 kb
EcoR I-
Xho I fragment from pCS-TPInt [
4], generating the plasmid pTP-I-hrGFP. A 1.4 kb
Eco RI fragment from pTA-
sre [
3], containing the R4 integrase, was cloned into the
Eco RI site of pIRES-hrGFP-1a, creating the expression plasmid pR4-I-hrGFP. The φC31 integrase was cloned from pCSI [
1] as a 1.9 kb blunted
Spe I-
Bam HI fragment and ligated into the
Sma I-
Bam HI sites of pIRES-hrGFP-1a, to generate the vector pφ-I-hrGFP. Each of these vectors expresses integrase under the control of the CMV promoter, followed by an internal ribosome entry site (IRES) and the humanized
Renilla reniformis GFP (hrGFP) open reading frame.
As a standard control for qPCR, the
att R junction for each integrase and a hygromycin gene region were cloned into a single plasmid. Vector p3BP was transformed into
E. coli strain DH-Int, which expresses the φC31 integrase [
1], in order to generate a φC31-excised plasmid. The
attR was PCR amplified and TOPO-cloned into vector pCR2.1 (Invitrogen, Carlsbad, CA), creating the plasmid pTA-φex. Next, the R4
attR was generated by intramolecular integration reaction in 293 cells co-transfected with p3BP and pCMV-sre [
3], creating p3BP-R4ex. The R4
attR junction was PCR amplified from p3BP-R4ex with primers that added
Spe I sites to each end of the
attR. The PCR product was digested with
Spe I and ligated into the
Spe I site of pTA-φex, creating plasmid pTA-φex-R4ex. The TP901-1
attR junction was generated by transforming p3BP into DH-TPInts [
4], generating plasmid p3BP-TPex. The TP901-1
attR was then PCR amplified, with addition of
Bam HI sites to each end. The PCR product was digested with
Bam HI and ligated into the
Bam HI site of pTA-φex-R4ex, creating the plasmid pTA-φex-R4ex-TPex. Next, a region of human
Rad52 gene was PCR amplified from 293 genomic DNA with primers that added
Cla I sites to each end. The PCR product was digested with
Cla I and ligated into the
Cla I site of pTA-φex-R4ex-TPex, creating the plasmid pTA-3attR-Rad52. Finally, a portion of the hygromycin gene encompassing the qPCR amplification region was PCR amplified from p3BP, digested with
Xba I, and ligated into the
Xba I site of pTA-3attR-Rad52, generating the standard vector p3attR-Rad52-Hyg. This vector was linearized with
Xcm I, purified, and serially diluted for use in qPCR to generate a standard curve.
Chromosomal deletion assay
293-3BP cell lines were transfected with 5 μg of pInt-I-hrGFP plasmid using Fugene 6 (Roche Applied Scientific, Indianapolis, IN), according to manufacturer's protocol. At 72 h post-transfection, GFP-positive cells were sorted and collected using a MoFLoPs FACS machine, thus sorting for cells that received integrase expression plasmid. Approximately 15 d after sorting and expansion, cells were harvested and genomic DNA was prepared using the Blood and Cell Culture DNA Maxi Kit (Qiagen, Valencia, CA).
Quantitative PCR analysis
Quantitative PCR on genomic DNA was performed using the following primer/probe combinations: TP901 attR: fwdTPex-qPCR 5'-TGATGTTACTGCTGATAATGTAGATATCATAT-3', revTPex-qPCR 5'-ATTAAAATTCACGGAAGAAAGCTTT-3', TPEx-attR-probe 5'-CGAGTTTTTATTTCGTTTATTTCAATCAAGGTAAATGC-3'; Φ C31 attR: fwdPhiEx-qPCR 5'-GGCTTCACGTTTTCCCAGGT-3', revPhiEx-qPCR 5'-CCAGATGGGTGAGGTGGAGT-3', PhiEx-attR-probe 5'-CTGGGGTAACCTTTGGGCTCCCCG-3'; R4 attR: fwdR4ex-qPCR 5'-TCTCATGCATAGAAGGCCCG-3', revR4ex-qPCR 5'-GGCTACACGGAGCAGGACC-3', R4ex-attR-probe 5'-CGATACCACTTGAAGCAGTGGTAGAAGGGCAC-3'. The hygromycin resistance gene was also amplified as an internal control for integrated p3BP vector, using the following primer/probe combination: fwdHyg-qPCR 5'-CTCGGAGGGCGAAGAATCTC-3', revHyg-qPCR 5'-GCAGCTATTTACCCGCAGGA-3', Hyg-probe 5'-TCAGCTTCGATGTAGGAGGGCGTGG-3'. Each of these probes was 5' labeled with the fluorophore 6-FAM and 3' labeled with the quencher TAMRA.
In addition to the primers and probes described above, PCR reagents used were from the TaqMan PCR Core Reagent Kit (Applied Biosystems, Foster City, CA). Amplification was performed in an ABI 7700 machine and the results were analyzed using SDS v1.7 software (Applied Biosystems). Each sample and standard was analyzed in triplicate.
Southern analysis
Genomic DNA was digested overnight with
Hin dIII. Twenty μg of digested genomic DNA was loaded onto a 0.6% TBE agarose gel and separated. The gel was depurinated in 0.25 M HCl, denatured in 0.5 M NaOH, neutralized in 0.5 M Tris-HCl (pH7.0), and transferred to an S&S Nytran blotting membrane (Schleicher & Shuell, Keene, NH) in 20X SSC transfer buffer. The membrane was probed with a 1.6 kb fragment from the hygromycin resistance gene or with a 1.6 kb genomic fragment from 8p22 encompassing φC31 human pseudo
attP site A (hpsA) [
2], both labeled with
32P by random primer extension, using the Ready-To-Go DNA Labeling Kit (-dCTP) (Amersham Pharmacia Biotech). Hybridization occurred at 65°C for ~20 h in Church buffer (1% BSA, 1 mM EDTA, 0.5 M NaHPO
4 pH 7.2, 7% SDS). Membranes were washed in 2x - 0.2x SSC at 65°C. Membranes were exposed to a phosphor screen and analyzed using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Southern blot quantification was performed using Kodak 1D software.