temperature gradient function of an iCycler (Bio-Rad) over 35 cycles. Subsequent experiments were carried out at the optimal Tm for each primer set in a GeneAmp PCR System 9600 (Applied Biosystems). Discrimination of 59 vs 39 NAIP transcript levels was carried out at 30 cycles. The full-length NAIP ORF deriving from the Alu SINEs was obtained by amplification with Phusion High Fidelity DNA Polymerase (Finnzymes). As expected, primers within Alu SINEs yielded a multitude of products and were subsequently resolved by Southern blotting. Probe was generated with radiolabeled dCTP32 using the random primer labeling kit (Invitrogen) as directed. Pre-hybridization, hybridization, and washes of Zeta-probe GT membranes (BioRad) were performed using ExpressHyb (Clontech) according to manufacturer’s speci- fications. Exposure of BioMax Film (Kodak) for one hour or less was sufficient to adequately differentiate true bands from background. 59 Rapid Amplification of cDNA Ends Using the First-choice RLM RACE Kit (Ambion) the 59 termini of human NAIP were deduced as before [15]. We revised our initial approach [15] by designing gene-specific reverse primers to a downstream exon, common to all predicted NAIP copies (primers listed in Table S1); previously primers could only surmise expression of NAIPfull. Subtle variations in RT-PCR product size was observed across a range of Tms (55u–60u) – since the full complement of NAIP start sites was being queried – therefore, all unique bands were purified using the QIAquick Gel Extraction Kit (Qiagen) and cloned into the pGEM-T vector (Promega) prior to sequencing (McGill University and Ge´nome Que´bec Innova- tion Centre). Importantly, consistent amplification patterns were observed within a given Tm. We similarly tested mouse kidney RNA; although we identified novel intraexonic start sites for mNaip2, qRT-PCR only showed a slight increase (1.2:1) of 39 over 59 ends (data not shown). Quantitative RT-PCR The cDNA used for quantitative RT-PCR with Power SYBR Green PCR Master Mix (Applied Biosystems) in the ABI 7500 Real Time PCR System (Applied Biosystems) was prepared as above. Primers (10 mM) were determined to amplify equally efficiently across a broad range of template dilutions by standard curve (listed in Table S1). The comparative CT method was used to quantify targets; CT values were normalized to b-actin levels in each tissue and expressed relative to the indicated target in the indicated tissues. Experiments were conducted at least four times for each primer set, with cycling parameters as follow: 50uC, 2 min; 95uC, 10 min; [95uC, 15 s; 60uC, 1 min] X 40 cycles. For initial experiments, where primer efficiencies were being deter- mined, dissociation curves and –RT controls were included, indicating the specificity of amplification and lack of DNA contamination in template preparations, respectively (data not shown). Alternative splicing variants posed a problem in primer design for the NAIPERV-P and NAIPSg targets. For NAIPERV-P we quantified only one of the variants and estimated that it accounted for ,40% of all total LTR-derived transcripts, as before [15]. For NAIPSg, we designed primers spanning exon junctions of both isoforms and combined their proportions. Generation of constructs Placental genomic DNA was obtained from the laboratory of Dr. P. Medstrand (Lund University) and subsequently used to PCR amplify the NAIP promoter regions and open reading frame (ORF). Promoter constructs. Testis-specific LTR (or NAIPERV-P), the ubiquitous NAIPfull, and the Alu-derived NAIPSg and NAIPJb Novel Promoters for Human NAIP promoters were amplified by PCR using Phusion High Fidelity DNA Polymerase (Finnzymes) in an iCycler (BioRad) over 35 cycles, the primers used are listed in Table S1. The respective products are approximately 500 bp and centered on the transcription start sites. All primers possessed BglII and HindII recognition sites to facilitate directional cloning into a modified pGL3B vector described elsewhere [15]. Sequencing (McGill University and Ge´nome Que´bec Innovation Centre) verified fidelity of amplified fragments. Expression vector. The preserved ORF deriving from NAIPSg and NAIPJb transcripts was amplified by Phusion High Fidelity DNA Polymerase (Finnzymes) from human testis cDNA (as described above) over 35 cycles, primer sequences are indicated in Table S1. The desired amplicon was isolated using the PureLink Quick Gel Extraction Kit (Invitrogen) and subsequently dATP- tailed with Taq DNA Polymerase (Invitrogen) to facilitate cloning into the pGEM-T vector (Promega). Sequencing not only confirmed that the ORF was cloned error-free, but also that NAIP2 is expressed, in addition to NAIPfull, on account of a single representative nucleotide difference. Xho1 and Nco1 recognition sites incorporated into primers were utilized to subclone the sequenced ORF into the CTV 211 hemagglutinin (HA) epitope- bearing mammalian expression vector, generously provided by Dr. R. Kay (Terry Fox Laboratory). All vectors were amplified in E. coli DH5a and purified using the Nucleobond AX (Clontech) maxi prep kit, and quantified using the Qubit fluorometer (Invitrogen). Cell culture and transient transfection HeLa, NTera2D1, LNCaP, and Jeg3 cells were cultured in DMEM (Stem Cell Technologies) and PC3 cells in RPMI 1640 (Stem Cell Technologies), and incubated at 37u and 5% CO2. All media formulations were supplemented with 10% Fetal Bovine Serum (Invitrogen) and maintained in penicillin/streptomycin, except when undergoing transfection experiments. Prior to transfection of promoter constructs cells were seeded at 105 cells/well, or 26105 cells/well for NTera2D1, in a 24-well dish overnight. Lipofectamine 2000 (Invitrogen) was used to transfect the indicated cells with the indicated vectors according to manufacturer’s specifications. Approximately 6-8 hours post- transfection cells were washed with PBS (Stem Cell Technologies) and fresh complete media was added to allow for production of the reporter for an additional ,24 hours. The HA:NAIP expression vector, was transiently transfected into HeLa, PC3, and NTera2D1 cells using Metafectene (Biontex) as recommended by the manufacturer. Reporter gene assays Prior to lysis, cells were washed with PBS, processed, then analyzed for firefly and Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega) as indicated by the manufacturer. All values were standardized to the Renilla luciferase internal control to normalize for transfection efficiency, then expressed relative to the modified promoterless pGL3-Basic vector. Western blotting Cells were grown in 10 cm dishes as indicated above. The human PC3, NTera2D1, and HeLa cell lines were selected to screen for NAIP proteins based on preliminary RT-PCR findings (data not shown). Cells transfected with the expression vector encoding the Alu-derived NAIP ORF or untransfected controls were harvested by either scraping or trypsinization following two washes with cold PBS. Cell pellets were obtained by centrifugation and resuspended in RIPA (150 mM NaCl; 1% NP-40; 0.5% PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e5761