Plasmids and vector construction.
The HSV vector D2 was created by genetic cross between the ICP4 deletion mutant virus d120 (5
) and an ICP27-deleted virus, 5dl1.2 (16
). D2 was identified based on its plaquing dependence on both ICP4 and ICP27 complementation and was confirmed by Southern blot analysis (not shown). BAC elements were engineered into the D2 genome at the thymidine kinase (TK) (UL
23) locus by homologous recombination in 7b cells and selection for ganciclovir resistance. The targeting plasmid for recombination was generated by insertion of the bacterial origin of replication and Cm resistance gene from pBeloBACII (New England Biolabs, Ipswich, MA) into the TK coding sequence of a UL
23 plasmid. The genome of a purified D2/BAC recombinant was circularized by infecting U2OS cells at a multiplicity of infection (MOI) of 5 for 3 h, and DNA was isolated by proteinase K digestion, phenol-chloroform extraction using PhaseLock gel (5Prime, Gaithersburg, MD), and isopropanol precipitation. The DNA was electroporated into GeneHogs bacteria (Invitrogen, Carlsbad, CA) at 2.0 kV, 200 Ω, and 25 μF in a 0.2-cm cuvette, and BAC DNA (DBAC) was purified from Cm-resistant bacteria using a large-construct DNA purification kit (Qiagen, Valencia, CA) with exonuclease digestion.
The Gateway cassette from Gateway conversion plasmid A (Invitrogen) was modified for this work in the following manner. First, the EcoRV fragment from plasmid A was cloned into the EcoRV site of pSP72 to generate p72GateA. p72GateA was modified to replace the Cm resistance gene in the Gateway cassette with phleomycin D (Zeocin [Zeo]) resistance by isolating and end filling an XhoI-EcoRI fragment containing the Zeo coding sequence from pEM7/Zeo (Invitrogen) and subcloning between the blunted NotI and MluI sites of p72GateA. Recombinants were screened for insertion in the opposite orientation to that of the ccdB gene to obtain plasmid p72GateAZ1. The NheI site between the attR1 and Zeor sequences was converted to a PmeI site by linker ligation to create plasmid pBZPme3. The modified Gateway cassette was isolated from pBZPme3 as a 1.6-kb EcoRV fragment containing attR1, a PmeI site, Zeor and ccdB genes, and attR2.
An ICP27-targeting plasmid containing the modified Gateway cassette was constructed in several steps. First, plasmid pHGatePme was created by cloning the EcoRV fragment from plasmid pBZPme3 into the PmeI site of plasmid pPme2 between the HCMV promoter and the simian virus 40 (SV40) poly(A) region. Plasmid pPme2 was derived from pEGFP-N1 (Clontech, Palo Alto, CA) by BamHI-BglII collapse, conversion of SspI to BglII and AseI to BglII by linker insertions, and replacement of the AgeI-NotI fragment with a PmeI linker. The modified Gateway cassette, along with the upstream promoter and downstream poly(A) region, was isolated from pHGatePme as a 2.3-kb BglII fragment and inserted into the BamHI site of plasmid pPXE to create the ICP27-targeting plasmid pPXE-HGate. Plasmid pPXE is pBluescript containing UL
54 (ICP27) flanks between EcoRI and XbaI restriction sites (18
). Functionality of pPXE-HGate with respect to the Gateway cloning system was confirmed using plasmid pENTR-gus and methods provided by Invitrogen.
The Gateway expression cassette from pPXE-HGate was introduced at the DBAC ICP27 locus by Red/ET recombination methods (GeneBridges GmbH, Heidelberg, Germany), as instructed by the manufacturer, in ccdB-resistant HerpesHogs bacteria. Recombinants were identified by bacterial selection for zeocin resistance and confirmed by field inversion gel electrophoresis (FIGE) analysis. A representative recombinant was designated DBAC-GW.
Generation of HerpesHogs.
In order to generate a ccdB-resistant bacterial strain with optimal characteristics for BAC DNA propagation and purification, we initially introduced the ccdB-containing plasmid pENTR-1A (Invitrogen) into GeneHogs bacteria (Invitrogen). Transformation of GeneHogs with pENTR-1A resulted in low numbers of kanamycin (Kan)-resistant colonies. Restriction analysis of plasmids from these colonies revealed three types of plasmids: those that were increased in size, those that were similar in size to pENTR-1A but displayed rearrangements, and approximately 10% that were similar to pENTR-1A. Further analysis of the plasmids of increased size identified a hot spot for transposon insertion within the ccdB gene inactivating the gene and thus allowing the plasmid to confer Kan resistance without a functional ccdB gene. The rearranged plasmids purified from pENTR-1A-transformed GeneHogs revealed a loss of ccdB function in each of 24 cases, as demonstrated by their ability to efficiently transform ccdB-sensitive DH5α bacteria to Kanr. A total of 48 plasmids that were indistinguishable from the parental plasmids were similarly assayed and yielded two that retained ccdB activity. Extensive attempts to cure these two ccdB-resistant bacterial strains of the pENTR-1A plasmid were unsuccessful, and we therefore developed the alternative strategy described below.
Plasmid pETRS was created by inserting the Cm and sacB genes into pENTR-1A, providing additional markers for positive (Cmr) and negative (5% sucrose sensitivity) selection, respectively. We mutagenized GeneHogs bacteria overnight with 10 μg/ml N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Sigma-Aldrich, St. Louis, MO), transformed the cells by electroporation with pETRS, and selected the bacteria for Cm resistance. Mutagenesis increased the transformation efficiency, presumably due to increased ccdB resistance frequency, by approximately 500-fold (D. Wolfe, unpublished observation). Colonies that tested positive for Kan resistance were then cured of the plasmid by selection on 5% sucrose. These cured strains were confirmed for sensitivity to Cm and Kan and then tested for ccdB resistance by transformation with pENTR-1A. The majority of the sucrose-selected colonies yielded abundant Kan-resistant transformants indicative of the acquisition of bacterial genome-borne ccdB resistance.
Bacterial growth and selection.
Yeast tryptone (YT) medium (2×) was used for growth of bacterial strains in liquid culture. Luria-Bertani (LB) broth with 1.3% agar was used for growth on solid support. Selective agents included ampicillin (200 μg/ml), chloramphenicol (12.4 μg/ml), kanamycin (40 μg/ml), zeocin (25 μg/ml), sucrose (5% [wt/vol]), and tetracycline (3 μg/ml). l-Arabinose (10% [wt/vol]) was used for induction of the pBAD promoter in the pRedET plasmid (GeneBridges). All bacterial strains were grown at 37°C except while performing GeneBridges recombineering, when 30°C was used to maintain the Red/ET temperature-sensitive Ori plasmid.
cDNA library construction in entry clones.
mRNA isolated from equal numbers of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells was converted to a cDNA library in a Gateway-compatible plasmid using the CloneMiner cDNA library construction kit (Invitrogen) as follows. Purified mRNA was reverse transcribed using SuperScriptII and a 5′biotin-attB2-oligo(dT) primer. After second-strand cDNA synthesis with Escherichia coli DNA polymerase I, T4 DNA polymerase was used to fill in the cDNA ends, and the products were collected by phenol-chloroform extraction and ethanol precipitation. A double-stranded attB1 adaptor was ligated to the 5′ end of the double-stranded cDNA with T4 DNA ligase at 16°C overnight. To favor the cloning of large inserts, the cDNA pool was size fractionated using a Sephacryl S-500 HR column. Adjacent fractions were combined and precipitated with ethanol. This final cDNA pool contained 5′ attB1 and 3′ attB2 linkers to allow for directional transfer into pDONR222 (Invitrogen), a plasmid containing Cmr and ccdB selectable marker genes between attP1 and attP2 sites and Kanr in the plasmid backbone.
Transfer of the cDNA into the donor plasmid was performed by incubating 250 ng pDONR222 and 150 ng of attB-flanked cDNA with BP Clonase enzyme mix (Invitrogen) at 25°C for 16 to 20 h. Following proteinase K-mediated inactivation of the enzyme mix, DNA was ethanol precipitated and electroporated into ccdB-sensitive E. coli DH10B. Electroporation parameters were 2.0 kV, 200 Ω, and 25 μF. The BP Clonase reaction promotes specific recombination between attB1 and attP1 sites and between attB2 and attP2 sites to create plasmid-based cDNA libraries flanked by 5′ attL1 and 3′ attL2 sites for subsequent LR Clonase-mediated transfer into the DBAC-GW destination vector. Plasmid DNA was isolated from 22 random Kan-resistant colonies and digested with BsrGI, which cuts in attL sites, to examine the efficiency of the BP Clonase reaction and the sizes of resulting plasmid inserts. Pooled transformants were grown in Kan-containing liquid media to an optical density at 600 nm (OD600) of 1.0.
Library introduction into HSV.
The cDNA library was transferred from its plasmid base into the DBAC-GW genome by LR Clonase (Invitrogen)-catalyzed recombination between the attL sites flanking the cDNA library and the attR sites flanking the Gateway cassette in the DBAC-GW destination vector, re-creating attB sites. The products of the LR recombination reaction were used to transform ccdB-sensitive DH10B cells, providing selection in the presence of Cm for loss of the ccdB gene from DBAC-GW in favor of library cDNAs. DBAC-L library DNA was purified from amplified transformants using a Qiagen large construct kit. To generate infectious virus, purified DBAC-L DNA was transfected into complementing 7b cells and the infection was allowed to progress to 100% cytopathic effect. Cells and supernatant were treated with NaCl (0.45 M final concentration), and the liquid fraction was aliquoted and stored at −80°C.
cDNA inserts were amplified from the plasmid-based entry library or the DBAC-L library using primers specific for the human cytomegalovirus (HCMV) promoter (GCGTGTACGGTGGGAGGTCTAT) and the SV40 polyadenylation region (GGGGAGGTGTGGGAGGTTTT) using Accuprime Taq polymerase (Invitrogen). The reactions were performed by a Bio-Rad iCycler IQ programmed as follows: 94°C for 3 min, 35 cycles of 94°C for 30 s, 61.7°C for 30 s, and 68°C for 1 or 5 min, and a final incubation at 68°C for 5 min. Approximately 100 ng of template DNA was used for each reaction. PCR products were purified using Qiaquick PCR purification kit (Qiagen) with an additional 80% ethanol wash prior to elution.
Microarray analysis of library inserts.
PCR products from each of four individual 1-min extension reactions were combined with PCR products generated by four 5-min extension reactions (see Fig. ) to create four pseudo-biological replicates. Approximately 10 μg of DNA was chemically labeled at guanine residues with Cy3 using the ULS labeling kit for Agilent gene expression arrays (catalog EA-023; Kreatech Biotechnology, Netherlands) according to the manufacturer's instructions with the following modifications: 10 μg of DNA was combined with 10 μl of Cy3 in a 50-μl reaction volume. Following the labeling reaction, each 50-μl reaction was purified on one Kreapure column according to the provided protocol. The labeled cDNAs were stored at −20°C in the dark prior to shipment to Cogenics, Inc. (Morrisville, NC), where the cDNA was fragmented and 3.3 μg of each labeled product was hybridized to an Agilent whole-rat genome oligonucleotide microarray in 4 × 44K format array. Hybridization, washing, staining, and scanning were conducted using established procedures at Cogenics.
FIG. 3. PCR amplification of DBAC-L cDNA inserts demonstrates library diversity. (A) cDNA inserts were amplified from DBAC-L genomic DNA, purified from E. coli, using primers specific for the HCMV promoter and SV40 poly(A) sequences. Extension times for PCRs (more ...)
A given feature (probe) on the microarray was determined to be “well above background” if the measured mean signal intensity for the given feature was significantly greater than the value for the corresponding background based on a two-sided t test and the background-subtracted signal for the feature was greater than 2.6 times the standard deviation of the measured background level. This approach enabled an array-by-array as well as a feature-by-feature determination of whether a given transcript was measured as “detected” in each sample for all of the noncontrol probes present on the array. The results were tabulated in order to determine which transcripts were identified as “detected” in all four samples, three out of four samples, two out of four samples, and one out of four samples, as well as not detected in any sample.
Gene expression microarray analysis.
Vero cells (2 × 106) were infected in duplicate with either the parental DBAC-GW vector, a characterized mix of 5 DBAC-L vectors (Mix5), or a mix of 100 randomly chosen DBAC-L viruses (Mix100) at a total MOI of 10. One day postinfection, total RNA was isolated using the Qiagen RNeasy kit and shipped to Cogenics for analysis.
Total RNA (500 ng) was converted into labeled cRNA using Cy3-coupled nucleotides and the low-RNA-input linear amplification kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer's instructions. Labeled cRNA samples (1.65 ng) were hybridized to Agilent whole-rat genome oligonucleotide microarrays (Cogenics). Hybridized arrays were washed and scanned, and the data were extracted using Feature Extraction software, version 9.1 (Agilent).
Pearson correlation values were calculated for all four pairs of biological replicate samples using all noncontrol features present on the microarray. A Pearson correlation value of 1.0 signifies perfect correlation, while 0 means no correlation and −1.0 denotes perfect anticorrelation. The replicates for each sample were then combined using an error-weighted average, and these combined profiles were compared to that of DBAC-GW-infected Vero cells in order to identify differentially expressed transcripts (threshold of 1.5-fold difference).
Western blot analysis.
Cells of the 7b line were infected at MOIs of 2 or 20 with viral vectors encoding tyrosine hydroxylase (TH) or succinate dehydrogenase subunit D (SDHD) isolated from the DBAC-L pool (unpublished results). Cell lysates from infected cells and PC12 control cells were harvested at 24 h postinfection (hpi) in 1× NuPage LDS buffer (Invitrogen), separated on a 4 to 12% SDS-PAGE gel (NuPage; Invitrogen), and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were reacted with antibodies to TH or SDHD (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxidase (HRP)-conjugated secondary antibodies.