Sequence information, maps, and material requests for these constructs can be found on our web site [70
- To generate a multiple cloning sequence flanked by FRT
recombinase recognition sequences (FRT
MCS), two oligonucleotides with overlapping sequence (shown in bold) were designed, FRT
], and FRT-loxP
]. These oligonucleotides were annealed and elongated by PCR using Pwo polymerase. The 218 base pair PCR fragment was cloned into pCR4 using the ZERO Blunt TOPO PCR Cloning Kit (Invitrogen, USA) to create pCR4 FRT
MCS, and its sequence was verified. FRT
MCS was subsequently excised with EagI and BspHI and cloned into pT2/BH [71
] cleaved with EagI and NcoI to produce pT2-FRT
MCS. Finally, a completely filled XhoI fragment, containing the mouse PGK promoter, the PTK fusion protein, and bovine growth hormone poly(A) signal from YTC37, a kind gift from the laboratory of A. Bradley [54
], was cloned into Sma1 cleaved pT2-FRT
MCS to produce pT2-FloxP-PTK.
- A 1.0 kb fragment of the SB11 transposase from pCMV-SB11 [72
], which had been amplified with CDS-SB11-F1 [CACCATGGGAAAATCAAAAGAAATCAGCC] and CDS-SB11-R1 [GGATCCCAATTTAAAGGCAATGCTACCAAATACTAG] primers and subcloned into an intermediate vector adding a 5' BglII site and the sequence [AGATCTGAT], was cloned into the BamHI site of pKUb to make pKUb-SB11. pKUb was made by cloning nucleotides 3561–4771 of the human UbC gene (genbank accession D63791
), which contains the UbC promoter, non-coding exon 1, and intron 1, into pK-SV40(A) between intact BglII and NheI restriction endonuclease sites. pK-SV40(A) was made by cloning a single copy of the SV40 poly(A) signal amplified by PCR with oligos KJC-SV40(A)-F1 [CATTGATGAGTTTGGACAAACCACA] and KJC-SV40(A)-R1 [ACCACATTTGTAGAGGTTTTACTTGCT] into pK-A10 opened with XmnI. pK-A10 was made by cloning KJC-Adapter 10 [CTGAGATCTTAAGCTAGCAGGATCCAGAATTCATTCAG] into pK digested with PvuII creating a multiple cloning site with PvuII, BglII, AflII, NheI, BamHI, EcoRI, XmnI, and PvuII recognition sites. pK was made by joining an 0.8 kb PCR product of pBluescriptSK- (Stratagene), containing the pUC_ORI amplified with oligos KJC-pUC_ORI-F1 [CTGTTCCGCTTCCTCGCTCACTGACT] and KJC-pUC_ORI-R1 [AAAAGGATCTAGGTGAAGATCCTTTTTGAT], to a 0.9 kb PCR product of pENTR-D-TOPO (Invitrogen), which contains the kanamycin resistance gene amplified by oligos KJC-KanR-F1 [CTGCATCATGAACAATAAAACTGTCTGCT] and KJC-KanR-R1 [TGCCAGTGTTACAACCAATTAACCAAT]. The junction of ORI-F1 to KanR-R1 created a single PvuII site.
pCMV-β is available from Clontech (Mountainview, CA).
pPGK(nls)CRE was a kind gift of Dr. David Largaespada's lab at the University of Minnesota.
pKT2P-(nls)FLP- A Flp open reading frame containing the large T antigen nuclear localization signal (bold) and a Kozak consensuses sequence was generated by amplifying the Flp open reading frame using primers CDS Kozak-NLS Flp 5' [ATATCTCGAGGCCACCATGGCTCCCAAGAAGAAGAGGAAGGTGATGAGTCAATTTGATATATTATGTAAAAC] and CDS Flp 3' [ATATAGATCTTTATATGCGTCTATTTATGTAGG] using pOG44 (Invitrogen, USA) as template. The resulting PCR product was cloned into pCR4 using the ZERO Blunt TOPO PCR Cloning Kit (Invitrogen, USA) creating pCR4-nlsFlp. The nlsFlp open reading frame was subsequently excised with XhoI and BglII and inserted into XhoI-BglII cleaved pKT2-PGKi to produce pKT2P-nlsFlp. pKT2-PGKi contains the human PGK promoter, a kind gift of Dr. Scott McIvor (University of Minnesota) in front of the mini-intron, MCS, and rabbit beta-globin 3'UTR found in mini-CAGs.
was made by cloning a 0.7 kb XhoI to BglII fragment of pKT2P-GeN into pKT2-mCAG opened from BglII to XhoI. pKT2-mCAG was made by cloning a 2.2 kb BamHI to KpnI fragment of pSBT-mCAG [73
] into pK-A3 opened from BamHI to KpnI. pKT2P-GeN was made by cloning EGFP as a 0.75 kb EcoRI fragment from pCR4-EGFP into the EcoRI site of pKT2P-eNeo. pCR4-EGFP was made by cloning a PCR fragment of EGFP from pEGFP-N1 (Clontech) amplified with primers KJC-EGFP-F3 [CCGAATTCTACCATGGTGAGCAAGGGCGAG] and KJC-EGFP-R2 [CCAGATCTTTACTTGTACAGCTCGTCCATGC] into pCR4-TOPO (Invitrogen). pKT2P-eNeo contains the encephalomyocarditis virus internal ribosome entry site and neomycin resistance gene amplified from pGT-Neo [62
] with KJC-BactinSA-F1 [CACTGAAGTGTTGACTTCCCTGACAGC] and KJC-Bgeo-R1 [TTCAATTGTTAGAAGAACTCGTCAAGAAGGCGA]. The eNeo cassette was subcloned and acquired a modified sequence at the 3' end [GTTAACTT] to [GTTAAGTCTAGA] including a BglII site. The 1.4 kb eNeo cassette was isolated with EcoRI and BglII and moved into pKT2-PGKi opened from BglII to EcoRI.
was made by cloning a 2.7 kb PvuII fragment from pKP-PTK_TS into pKT2-RV opened with EcoRV. pKT2-RV was made by cloning a 0.6 kb BamHI to KpnI fragment of pSBT-RV [73
] into pK-A3 opened with BamHI and KpnI. pK-A3 was made by opening pK with PvuII and inserting KJC-Adapter 3 [CTGGATCCAGATCTGGTACCATTTAAAT] creating a small multiple cloning site with PvuII, BamHI, BglII, KpnI, and SwaI sites. pKP-PTK_TS was made by cloning a 2.3 kb BglII to EcoRI fragment of pCR4-PGK-PTK into the MCS of pK-SV40(×2) opened with EcoRI and BglII. pCR4-PGK-PTK was made by cloning a 2.3 kb PCR product of pT2-FloxP-PTK amplified with PuroΔTK-F1 [TTAGATCTGGCCTCGCACACATTCCACAT] and PuroΔTK-R1 [TGGTTCTTTCCGCCTCAGAAGCCAT] into pCR4-TOPO (Invitrogen). pK-SV40(×2) was made by cloning two copies of the SV40 poly(A) signal amplified by PCR with oligos KJC-SV40(A)-F1 [CATTGATGAGTTTGGACAAACCACA] and KJC-SV40(A)-R1 [ACCACATTTGTAGAGGTTTTACTTGCT] into pK-A10 opened with XmnI.
- The mini Tol2
transposon donor plasmid was constructed by inserting the PvuII fragment of pKP-PTK-TS into pGemT-Tol2 [74
] opened from SwaI to HindIII (filled) to produce pGTol2P_PTK.
was constructed as indicated [74
] from previously described materials [75
was made by cloning a 2.7 kb PvuII fragment of pKP-PTK_TS into pPBT-SE opened from SmaI to EcoRV. pPBT-SE was made by cloning the 102 bp PCR product containing an outward facing T7 polymerase site, the SE multiple cloning site, and an outward facing T3 polymerase site into pPBT cut with MscI. The PCR product was amplified from pKT2-SE using T7-RevComp [TCTCCCTATAGTGAGTCGTATTA] and T3-RevComp [TCTCCCTTTAGTGAGGGTTAATT] primers. pPBT was made by cloning the PB LTR1 and LTR2 into pKT2-SE from KpnI to BamHI. LTR1 and LTR 2 from PB were amplified from pXL-Bac-II, a kind gift of Malcolm Fraser (Notre Dame University), using PB-LTR1-F1 [TGGATCCCAATCCTTAACCCTAGAAAGATAATCATATTG] and PB-LTR1-R1 [GTGGCCATAAAAGTTTTGTTACTTTATAGAAG] or PB-LTR2-F1 [TTGGCCATAAGTTATCACGTAAGTAGAACATG] and PB-LTR2-R1 [TGGTACCTAGATTAACCCTAGAAAGATAGTCTG], respectively. LTR1 and LTR2 PCR products were cloned into pCR4 vector (Invitrogen) and subsequently excised by BamHI and MscI or MscI and KpnI digestion, respectively. pKT2-SE was made by cloning the 0.7 kb BamHI to KpnI fragment containing the SB inverted repeats and SE multiple cloning site from pSBT-SE [73
] into pK-A3 opened from KpnI to BamHI.
pKC-PB was made by inserting the 2.1 kb NheI to BamHI fragment of p3XP3-DsRed, a kind gift of Dr. Malcolm Fraser (Notre Dame University), containing the PB transposase coding sequence into the 3.2 kb BamHI to NheI fragment of pKC-SB11, which resulted in the exchange of SB11 with PB transposase.
- A 2.7 kb PvuII to PvuII fragment of pKP-PTK_TS was cloned into the EcoRV site of pPTn2-RV to make pPTnP-PTK. pPTn2-RV was made by cloning KJC-Adapter 4 [TCTCCCTTTAGTGAGGGTTAATTGATATCTAATACGACTCACTATAGGGAGA] into the MscI site of prePTn2(-1) creating T7 and T3 polymerase binding sites orientated out towards the inverted repeats of the PTn transposon and separated by an EcoRV site. prePPTn2(-1) was made by cloning a 0.5 kb BamHI to KpnI fragment of pCR4-PPTN2A into pK-A3 opened from KpnI to BamHI. pCR4-PPTN2A was created by topo cloning a 0.5 kb PCR product amplified from prePPTN2(-2) using oligos PPTN-F1 (BamHI) [AAGGATCCGATTACAGTGCCTTGCATAAGTAT] and PPTN-R1 (KpnI) [AAGGTACCGATTACAGTGCCTTGCATAAGTATTC] into pCR4-Topo (Invitrogen). prePPTN2(-2) was created by amplifying the majority of pBluKS-PPTN5 [29
], a kind gift of Dr. Michael Leaver (University of Stirling, UK), with oligos PPTN-OL2 [CCATCTTTGTTAGGGGTTTCACAGTA] and PPTN-OR1 [CCAGGTTCTACCAAGTATTGACACA]. The PCR fragment was then self-ligated to produce an empty transposon with a single MscI site in its interior.
was made by cloning a 1.0 kb NheI to EcoRI fragment of pKUb-PTs1 that contained the PPTN transposase (PTs) into pK-mCAG opened from EcoRI to NheI. pK-mCAG was made by cloning the mCAG promoter from pSBT-mCAG [73
] as a 0.96 kb SmaI to EcoRI (filled) fragment into pK-SV40(A) × 2 opened with AflII (filled). pKUb-PTs1 was made by replacing the SB11 gene from pKUb-SB11 with PTs by cloning a 1.0 kb BamHI to NheI fragment from pCR4-PPTs1B into pKUb-SB11 from NheI to BamHI. pCR4-PPTs1B was made by cloning a PCR fragment of pBluKS-PPTN4 [29
], a kind gift of Dr. Michael Leaver (University of Stirling, UK), amplified with primers CDS-PPTs-F1 [AAAGCTAGCATGAAGACCAAGGAGCTCACC] and CDS-PPTs-R1 [AAGGATCCTCAATACTTGGTAGAACC] into pCR4-Topo (Invitrogen).
pKT2C-loxPTK-G was made by cloning a 2.3 kb PvuII fragment of pK-PTK_TS into the MscI site of pKT2C-lox-GFP. pK-PTK_TS was made by cloning a 1.9 kb BglII to EcoRI fragment of pCR4-PTK into the MCS of pK-SV40(×2) opened with EcoRI and BglII. pCR4-PTK was made by cloning a 1.9 kb PCR product of pT2-FloxP-PTK using oligos PuroΔTK-F2 [TTAGATCTACCATGACCGAGTACAAGCCCA] and PuroΔTK-R1 [TGGTTCTTTCCGCCTCAGAAGCCAT] into pCR4-TOPO (Invitrogen). pKT2C-lox-GFP was made by cloning 0.1 kb EcoRI fragment of pCR4-loxP, which contains two direct repeat loxP sites separated with a MscI site, into pKT2C-EGFP opened with EcoRI. pCR4-loxP was made by topo cloning the annealed and extended oligos loxP-F1 [ATAACTTCGTATAATGTATGCTATACGAAGTTATCTCGAGTGGCCA] and loxP-R1 [ATAACTTCGTATAGCATACATTATACGAAGTTATTGGCCACTCGAG] into pCR4-TOPO (Invitrogen).
Cell Culture and transposition/recombinase assays
Pig fibroblasts were isolated from 43 day old embryos. The tissue was dissociated using a collagenase/DNAse I treatment as well as mechanical disruption. The cells from the female piglet #8 were cultured in DMEM enriched with 10%FBS and 2× antibiotic/antimycotic solution (Gibco #15240-022). The cells were passaged in DMEM high glucose media enriched with 10% FBS, 2 mm L-glutamine, 1× P/S until spontaneously establishing line PF8. A subpopulation of porcine endometrial gland epithelium cells [76
] were spontaneously immortalized, strain PEGE. The PEGE cells were maintained in DMEM supplemented with 10% FCS, 1× Penn/Strep, 10 μg/ml Insulin (Sigma, USA), and 1× L-Glutamine.
For transposition assays cells were plated in each well of a six well plate to achieve 60–80% confluence within 6–24 hours. Cells were transfected using Trans
IT-LT1 (Mirus Bio Corporation, WI) transfection reagent according to the manufactures instructions with a ratio of 3:1 lipid: μg DNA. Each transfection contained a total of 1.15 to 1.5 μg of plasmid DNA. Wells 1–3 contained transposon plus transposase, well 4 contained transposon with no transposase, well 5 contained SB plus SB transposase and well 6 contained pKT2C-EGFP only. Molar amounts of each transposon were fixed at 1.5 × 10-13
moles of transposon (0.75 × 10-13
Moles for Tol2
) while transposase plasmid was added at a molar ratio of 1:1 for SB, Tol2
, and PB
, and 1:0.5 for PP. The choice of the promoters and transfection ratios for SB and PP was based on the highest transposition activity observed in human HT1080 cells (data not shown). Strong promoters (CMV & miniCAGs) and transfection conditions for Tol2
and PB were selected based on previously published data and the observation that these transposon systems seem less susceptible to overexpression inhibition than SB and PP.[34
] Total DNA weight was adjusted using pCMV-β plasmid. Forty-eight hours after transfection cells were trypsinized, and two replicates of 60,000 cells were plated onto 100 mm plates in media containing 0.3 μg/ml puromycin and selected for 9–12 days. Colonies were visualized by methylene blue staining and counted. A minimum of two six-well plates were transfected for each experiment. The mean colony number and standard error are shown in figures.