linear puromycin marker takara bio  (TaKaRa)

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: Exon junctions from RNA sequencing data of three different HPV49 E8- genome containing cell lines were identified (HISAT2 aligner, enable spliced alignment), displayed and counted with the integrated genome viewer, and are depicted as a Sashimi plot. Read abundances on the y-axis are shown on a linear scale. Only splice junctions with >100 supporting reads are shown. Only splice junctions with more than 100 reads and present in all three cell lines are listed in the table. The linearized HPV49 reference genome with ORFs is shown below. The positions of consensus poly adenylation sites are indicated by pins.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: RNA Sequencing Assay

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: ( A ) Agarose gel analysis of the RT-qPCR product ( P ) to detect the SD217/SA3281 splice junction in total RNA isolated from HPV49 E8- cells. A size marker ( M ) is shown on the left. A partial sequence of the RT-qPCR product obtained is shown below. qPCR analysis of unspliced E6 and spliced SD217/SA3281 transcripts in stable HPV49 E8- cell lines using total RNA ( B ) or in NHK transiently transfected with HPV49 wt or E8- genomes harvested 3, 6, or 9 days post transfection using polyA + -enriched RNA ( C ). ( B ) n = 9, paired t-test. (*** P < 0.001); ( C ) n = 4. Error bars indicate the SEM.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: Agarose Gel Electrophoresis, Quantitative RT-PCR, Isolation, Marker, Sequencing, Transfection

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: ( A ) Sequence alignment of the SD consensus , HPV49 SD217 wt, SD217 mt, and codon-optimized (co) E6 sequences. ( B ) qPCR analysis of unspliced E6 transcripts using polyA + -enriched RNA isolated from C33A cells transfected with pSG HPV49 3xHA E6 ( E6 ), pSG HPV49 3xHA E6 SD217 mt (E6 SD217 mt), or pSG HPV49 3xHA E6co (E6 co) plasmids. n = 5, statistical significance was determined by a ratio-paired t-test (*** P < 0.01). ( C ) Immunoblot analysis of C33A (left panel) or NHK (right panel) transfected with the empty vector or expression vectors for 3xHA-tagged wt E6, E6 co, or E6 SD217 mt is shown below. E6 was detected with an anti-HA antibody, and HSP90 was used as a loading control. In the right panel, a pre-stained protein marker ( M ) is shown.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: Sequencing, Isolation, Transfection, Western Blot, Plasmid Preparation, Expressing, Control, Staining, Marker

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: ( A ) Overview of the immortalization capabilities of HPV49 E6 and E7 in NHK. NHK from three different donors were transduced with combinations of recombinant retroviruses as indicated in the table. + indicates immortalization, +/- indicates prolonged life span, and - indicates no prolonged life span. ( B ) Growth curves of cell lines immortalized with HPV49 E7 and with or without different E6 (wt, co, and SD217 mt). Data are derived from five to nine independent experiments for E6/E7 cell lines. The growth curve of HPV49 E7 is derived from one experiment. Error bars indicate the SEM.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: Transduction, Recombinant, Derivative Assay

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: NHK immortalization assays with different HPV49 genomes

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques:

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: Viral gene expression analysis of NHK transiently transfected with different HPV49 genomes as indicated by qPCR using PGK1 as a reference gene. ( A ) PolyA + -enriched RNA was isolated 6 days post transfection and analyzed for SD217/SA3281 , E6 , E1 , and E7 transcripts. ( B ) Total RNA was isolated 6 days post transfection and analyzed for URR^E4 , E8^E2 , E1^E2 , and E1^E4 transcripts. Values were calculated from plasmid standard curves. Data are derived from five ( A ) or nine ( B ) independent transfection experiments. Statistical significance was determined using a ratio-paired t-test using wt/ E8- as reference for the respective SD217 mt (* P < 0.05). Error bars indicate the SEM.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: Expressing, Transfection, Isolation, Plasmid Preparation, Derivative Assay

Journal: Journal of Virology

Article Title: A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

doi: 10.1128/jvi.01640-24

Figure Lengend Snippet: Transcript map of HPV49. The HPV49 genome was linearized at nt. 7,500 to enable the depiction of transcripts initiated at the putative promoter in the 5′URR. ORFs are shown above the linearized genome. Potential TSS are depicted by arrows and polyadenylation signals by pins. Spliced transcripts are shown below the linearized genome. Solid lines represent introns removed from spliced transcripts and dotted lines indicate unknown 5′- or 3′-extensions. Numbers indicate the last or first nt. of the exon.

Article Snippet: PMSCV-puro HPV49 E7 is based upon pMSCV-puro (Clontech), and the HPV49 E7 sequence was inserted between the BglII and EcoRI restriction sites.

Techniques: