Improvement in the design of MMTV LTR-based transfer vectors and their test in the <i>in vivo</i> packaging assay.

<p>(<b>A</b>) Schematic representation of the second set of MMTV transfer vectors in which the U3 region containing the promoter within the 5′ LTR was replaced with the promoter sequences of human cytomegalovirus (hCMV) generating a chimeric CMV-R/U5 LTR. In addition, the constitutive transport element (CTE) from the Mason-Pfizer monkey virus (MPMV) was inserted between the Δ<i>env</i> and the 3′ LTR to facilitate efficient nuclear export of viral genomic RNA in the absence of an intact MMTV Rem/RmRE export pathway. (<b>B</b>) Schematic representation of the three series of transfer vectors tested in the study containing chimeric 5′ MMTV LTR and therefore following transfection the RNA transcription was initiated by the hCMV promoter sequences. The first series of transfer vectors (DA19–24) tested the entire 5′ UTR in the presence of incremental amounts of <i>gag</i> sequences, the second series (FA21–26) tested only incremental amounts of 5′ UTR sequences in the absence of any <i>gag</i> sequences, while the third series (NS07–12) tested the same incremental amounts of 5′ UTR sequences in the presence of 400 nt of <i>gag</i>. (<b>C</b>) RT-PCR of cytoplasmic RNA fractions to ensure the integrity of the fractionation technique and conventional PCR on RNA samples to monitor the absence of any contaminating DNA in the RNA preparations. Panel I: Multiplex PCR amplification of cytoplasmic cDNAs with 18S and unspliced ß-actin primers; Panel II: PCR amplification of cytoplasmic cDNAs with spliced ß-actin primers; Panel III: PCR amplification of DNase-treated RNA with GAPDH primers. Lane numbers 1–18 correspond to the numbers of the three series of chimeric LTR transfer vectors (DA, FA, and NS) in sequential order, whereas lanes 19 and 20 correspond to cytoplasmic fraction from Mock (containing the packaging construct, JA10 + the VSV-G-Env expression plasmid, MD.G + luciferase-expression vector, pGL3) and no DNA transfected cultures. +C, positive control (cDNA from cellular mRNA); M, molecular weight markers.</p

Relative expression of transfer vector RNAs within the transfected cells.

<p>Real time PCR analysis of (<b>A</b>) cytoplasmic transfer vector RNAs and (<b>B</b>) cytoplasmic β-actin RNA expression expressed as logΔRn verses cycle number. ΔRn is the target gene-specific fluorescence signal (FAM for MMTV-specific and VIC for ß-actin-specific sequences) normalized to the signal for the internal passive control, ROX (Normalized Reporter or Rn) from which the baseline target fluorescence has been subtracted (ΔRn = Normalized Reporter (Rn) - baseline). (<b>C</b>) Relative cytoplasmic transfer vector RNA expression in 293T cells after normalization with β-actin and luciferase expression. MK, Mock, transfected cultures with packaging construct, JA10 + the VSV-G-Env expression plasmid, MD.G + luciferase-expression vector, pGL3 except the transfer vector. RQ, Relative Quantification in log₁₀ units. The primers for detecting the transfer vectors were designed within the U5 region of the MMTV LTR, a region common to all transfer vector RNAs. Each sample was tested in duplicates with MMTV- and β-actin-specific probes and primers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#s4" target="_blank">Materials and Methods</a>.</p

Validation of the real time PCR assay developed for relative quantification of transfer vector RNA expression using the Ct slope method.

<p>Determination of the threshold cycles (Ct) of the cDNA prepared from 293T cytoplasmic RNA expressing transfer vector DA24 tested in triplicates as 10- and 2-fold dilutions by the (<b>A</b>) custom-made MMTV Taqman assay, and (<b>B</b>) the ß-actin Taqman assay. ΔRn is the target gene-specific fluorescence signal (FAM for MMTV-specific and VIC for ß-actin-specific sequences) normalized to the signal for the internal passive control, ROX (Normalized Reporter or Rn) from which the baseline target fluorescence has been subtracted (ΔRn = Normalized Reporter (Rn) - baseline). Standard curves of the (<b>C</b>) MMTV and (<b>D</b>) ß-actin Taqman assays were generated to determine their ΔCt values (Ct values of the MMTV assay – Ct value of the ß-actin endogenous control) that were needed to allow comparison of amplification efficiencies of the two assays. (<b>E</b>) Relative amplification efficiencies of the two assays as determined by the analysis of ΔCt value variations with the amount of input template cDNAs. For the two assays to have similar amplification efficiencies, the value of the slope of log input amount verses ΔCT should be approximately zero (<0.1), which under our experimental conditions were calculated to be 0.0126, thus validating the assay for the relative quantification analysis.</p

Propagation efficiency of MMTV transfer vector RNAs containing chimeric 5′ LTRs.

*<p>JA10, MMTV gag/pol packaging expression vector; MD.G, vesicular stomatitis virus (VSV-G) envelope expression vector; pGL3, luciferase expression vector.</p>**<p>Propagation of the transfer vector RNA expressed as hygromycin resistance colony forming units (CFU)/ml of viral supernatant that was used to infect target cells. The data represents the mean of at least three independent transfection and infection experiments testing all mutants and was derived after normalization to the transfection efficiencies observed by luciferase expression from a co-transfected luciferase expression vector. SD, standard deviation.</p>***<p>Entire UTR refers to 160 bp excluding 17 bp of primer binding site (PBS).</p>****<p>The differences observed in the RNA propagation abilities of DA24 and NS12, both containing same amounts of 5′ UTR and gag sequences could be attributed to an artificially introduced <i>Spe</i>I site in NS12 at the junction of 5′ UTR and gag during cloning. This <i>Spe</i>I site may have destabilized some sequence/structural motifs important for MMTV transfer vector RNA packaging and propagation (compare DA24 and NS12 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#pone-0047088-g005" target="_blank">Figure 5C</a>).</p

Experimental design to determine the role of the 5′ untranslated region (UTR) and <i>gag</i> sequences in MMTV RNA packaging.

<p>(<b>A</b>) Schematic representations of the complete MMTV genome and the MMTV long terminal repeats (LTRs)-based transfer vectors used as RNA packaging substrates in the study. In these transfer vectors, the wild type (WT) MMTV LTRs were maintained and therefore following transfection the RNA transcription was initiated by the promoter sequences within the U3 region of the 5′ LTR. Region encompassing most of the structural and enzymatic genes (<i>gag</i>, <i>pro, pol</i>, part of <i>env</i> and <i>rem</i>) were replaced by the SV-40 <i>hygromycin</i> resistance gene cassette as a marker for successful transduction of the target cells by packaged transfer vector RNA. (<b>B</b>) Three series of deletion mutations at the 5′ end of MMTV transfer vector sequences were introduced to monitor their effect on RNA packaging and propagation. The first series of deletion mutants (DA07–12) contained the entire 5′ UTR in the presence of incremental amounts of <i>gag</i> sequences, the second series of mutants (FA07–12) contained deletions in the 5′ untranslated region (UTR) sequences in the absence of any <i>gag</i> sequences, while the third series of mutants (NS01–06) contained the same incremental amounts of 5′ UTR sequences in the presence of 400 nucleotides (nt) of <i>gag</i>. (<b>C</b>) Table showing viral titers observed post transduction of HeLaT4 target cells by transfer vector RNAs tested. None of the transfer vector RNAs containing deletions in the 5′ UTR in the absence (FA07–FA12) or the presence (NS01–NS05) of <i>gag</i> sequences could be propagated (<1 CFU/ml) except NS06. *JA10, MMTV packaging Gag/Pol expression construct; MD.G, vesicular stomatitis virus (VSV-G) Env expression plasmid; pGL3, luciferase expression plasmid. **Propagation of the transfer vector RNA expressed as hygromycin resistance colony forming units (CFU)/ml of viral supernatant that was used to infect target cells. ***The entire UTR refers to 160 nt excluding 17 nt of primer binding site (PBS). The data represents mean of at least three independent transfection and infection experiments testing all mutants and was derived after normalization to the transfection efficiencies observed by luciferase expression from a co-transfected luciferase expression vector. SD, standard deviation. (<b>D</b>) Reverse transcriptase (RT) PCR analysis of the MMTV LTR-based DA series of transfer vectors containing incremental amounts of <i>gag</i> sequences in the presence of the entire 5′ UTR followed by Southern blotting. The probe was prepared by PCR amplification of a 142 nt long R/UR/5′ UTR region (nt 1179–1321) common to all the transfer vectors, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#s4" target="_blank">Materials and Methods</a>. Amplification was carried out for either 25 (lower panel) or 30 cycles (upper panel) using transfer vector-specific primers OTR671 and OTR 672.</p

Relative packaging efficiency of MMTV transfer vector RNAs into the pseudotyped MMTV particles.

<p>Real time PCR analysis of (<b>A</b>) packaged β-actin RNA into VSV-G-Env-pseudotyped MMTV particles and (<b>B</b>) packaged transfer vector RNAs into the VSV-G-Env-pseudotyped MMTV particles expressed as ΔRn verses cycle number. ΔRn is the target gene-specific fluorescence signal (FAM for MMTV-specific and VIC for ß-actin-specific sequences) normalized to the signal for the internal passive control, ROX (Normalized Reporter or Rn) from which the baseline target fluorescence has been subtracted (ΔRn = Normalized Reporter (Rn) - baseline). (<b>C</b>) Relative RNA packaging efficiencies for each of the mutant transfer vector RNA after normalization with β-actin and luciferase expression. The box in panel B highlights the wide range of threshold cycle (Ct) values observed for each transfer vector in comparison to the similar amounts of β-actin packaged into the viral particles. The arrow in panel C highlights the threshold value of detection. MK, Mock, cells transfected with packaging construct, JA10 + the VSV-G-Env expression plasmid, MD.G + luciferase-expression vector, pGL3 except the transfer vector. RQ, Relative Quantification in log₁₀ units. *, statistically significant differences between constructs are shown by the brackets (p<0.01). #, statistically significant difference between NS12 and DA24 (p<0.01). The primers/probe for detecting the transfer vectors were designed within the U5 region of the MMTV LTR, a region common to all transfer vector RNAs. Each sample was tested in triplicates with MMTV- and β-actin-specific probes and primers in panel B, while the β-actin samples shown in panel A were tested in duplicates, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047088#s4" target="_blank">Materials and Methods</a>.</p