Difference between revisions of "Part:BBa K4218004"
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===Characterization=== | ===Characterization=== | ||
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| + | BBa_K4218004</span> | ||
| + | </p> | ||
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| + | <span style="font-family:'Times New Roman'"> | ||
| + | Characterization </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
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| + | <img src="./04_files/aratn-rw9xo.001.png" width="522" height="211" alt="IMG_256" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
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| + | <span style="font-family:'Times New Roman'"> | ||
| + | The dual luciferase reporter system</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | A previous study indicated exons of MAP3K7 were skipped in MDS patients. In order to detect the dysregulation of RNA splicing, we designed two exon-skipping LUC reporters (MAP3K7-LUC) which contains parts of the MAP3K7 intronic and exonic sequences in the luciferase gene(more details about the principle were listed in the project description). </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:14pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | Results</span> | ||
| + | </p> | ||
| + | <ol type="1" style="margin:0pt; padding-left:0pt"> | ||
| + | <li style="margin-left:14pt; text-align:justify; widows:0; orphans:0; padding-left:4pt; font-family:'Times New Roman'; font-size:12pt; font-weight:bold"> | ||
| + | <a name="OLE_LINK12"> | ||
| + | <span> | ||
| + | Construction of MAP3K7-LUC</span> | ||
| + | </a> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK12"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | The plasmid of MAP3K7-LUC was synthesized by Nanjing Genscript Biotechnology Corporation. The map of the plasmid was listed below (Fig 1). When we got the plasmid, we transformed it into DH5α bacteria and screened the positive clones on LB solid plates containing ampicillin (Fig 2). To test the quality of the plasmid, MAP3K7-LUC was analyzed on 1% Agarose Gel (Fig 3). Moreover, MAP3K7-LUC was also confirmed by using sequencing (Fig 4).</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.002.png" width="323" height="262" alt="" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 1. Map of the pGL-duel plasmid vector containing parts of the MAP3K7 intronic and exonic sequences </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.003.png" width="322" height="245" alt="修改图_01" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:7.5pt; text-align:center; line-height:18pt; widows:0; orphans:0; background-color:#ffffff"> | ||
| + | <span style="font-family:'Times New Roman'; font-size:10.5pt"> | ||
| + | Fig 2. Solid plates of MAP3K7-LUC sensor </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.004.png" width="285" height="224" alt="修改图_02" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <a name="OLE_LINK10"> | ||
| + | </a> | ||
| + | <a name="OLE_LINK13"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </a> | ||
| + | <a name="OLE_LINK15"> | ||
| + | </a> | ||
| + | <a name="OLE_LINK14"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 3. Electrophoresis of MAP3K7-LUC sensor (1)).</span> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK15"> | ||
| + | </span> | ||
| + | </a> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK10"> | ||
| + | </span> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK13"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.005.png" width="449" height="140" alt="" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 4. The MAP3K7-LUC plasmid sequencing</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | 1.</span> | ||
| + | <span style="font:7pt 'Times New Roman'"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | Functional verification of the MAP3K7-LUC plasmid sensor</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | In order to test the ability of MAP3K7-LUC to detect dysregulation of RNA splicing, </span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | Pladienolide B (PB, an RNA splicing inhibitor) was used. The plasmid </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC was transfected into 293T cells, </span> | ||
| + | <a href="javascript:;" style="text-decoration:none"> | ||
| + | <span style="font-family:'Times New Roman'; color:#000000"> | ||
| + | respectively</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | . </span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | PB (final concentration: </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1 ng/μl</span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | ) was added to disturb the process of RNA splicing. According to the principle of our plasmid sensor, the fusion proteins (Fluc-Rluc) were expressed in 293T cells. However, when RNA splicing was interrupted, only the Fluc proteins were induced</span> | ||
| + | <a name="OLE_LINK11"> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | (more details about the principle were listed in the project description)</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | . After transfection, cells were lysed and the expression of luciferase was measured by using plate reader (SpectraMax</span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | i3). In normal 293T cells, the expression of the fusion proteins (Fluc-Rluc) was high (Table 1, Fig 5). PB treatment induced the downregulation of the fusion proteins (Table 1, Fig 5). </span> | ||
| + | <a name="OLE_LINK2"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | The ratio of </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+ Fluc</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | )</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | to Rluc intensity [</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+Fluc</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | )</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | /Rluc]</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | was significantly decreased in cells treated with PB, compared with normal cells </span> | ||
| + | <a name="OLE_LINK6"> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | (Table 1, Fig 5)</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | . These results suggested </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC </span> | ||
| + | <span style="font-family:'Times New Roman'; background-color:#fcfdfe"> | ||
| + | sensor can detect the alteration of RNA splicing in cells. </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <a name="OLE_LINK1"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Table 1. The value of fluorescence in cell transfected with MAP3K7-LUC</span> | ||
| + | </a> | ||
| + | </p> | ||
| + | <table cellspacing="0" cellpadding="0" style="width:97.3%; border-collapse:collapse"> | ||
| + | <tbody> | ||
| + | <tr style="height:31.65pt"> | ||
| + | <td style="width:23.86%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:11pt"> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK1"> | ||
| + | </span> | ||
| + | <a name="_Hlk116131814"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </a> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+Fluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (Rluc+Fluc)/Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td style="width:23.86%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Blank</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 95</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 88</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1.08 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td rowspan="3" style="width:23.86%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 26915296</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 20458</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1315.00 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 11016970</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 8401</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1311.00 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 20162960</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 18755</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1075.00 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td rowspan="3" style="width:23.86%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC+PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 7955001</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 10674</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 745.21 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 7454314</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 9817</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 759.27 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.1pt"> | ||
| + | <td style="width:27.24%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 11537627</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:23.38%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 19522</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:25.48%; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 590.98 </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10pt"> | ||
| + | <span style="-aw-bookmark-end:_Hlk116131814"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.006.png" width="197" height="261" alt="演示文稿1_01" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | Fig 5. The relative luciferase activity in cells. </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Cells were transfected with MAP3k7-LUC treated with PB for 48 h. </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold; color:#2a2b2e"> | ||
| + | 2.</span> | ||
| + | <span style="font:7pt 'Times New Roman'"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | The sensitivity of </span> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | MAP3K7-LUC </span> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | sensor</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | In order to test the sensitivity of MAP3K7-LUC sensor, different concentrations of plasmid (final concentration: </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1 ng/μl</span> | ||
| + | <span style="font-family:'华光幼线_CNKI'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | 、</span> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | 0.6 </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | ng/μl</span> | ||
| + | <span style="font-family:'等线'"> | ||
| + | 、</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.2 ng/μl</span> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | ) were transfected into cells. PB treatment decreased the ratio of</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+ Fluc</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | )</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | to Rluc intensity [</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+Fluc</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | )</span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | /Rluc]</span> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | in cells transfected with MAP3K7-LUC plasmid. Our data indicated even a small number of plasmid sensors can detect the changes of intracellular splicing, suggesting the sensitivity of our sensors is high (Table 2 and , Fig 6).</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Table 2. The value of fluorescence in cell transfected different concentrations of MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | <div style="text-align:center"> | ||
| + | <table cellspacing="0" cellpadding="0" style="width:417.15pt; margin-right:auto; margin-left:auto; border-collapse:collapse"> | ||
| + | <tbody> | ||
| + | <tr style="height:31.05pt"> | ||
| + | <td style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+Fluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (Rluc+Fluc)/Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Blank</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 83</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 76</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1.09</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td rowspan="3" style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.3μg MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 8195064</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 14814</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 553.19</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 7980109</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 13543</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 589.23</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 5098527</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 8352</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 610.43</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td rowspan="3" style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.3 μg MAP3K7-LUC+PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1671028</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 6201</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 269.46</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1424451</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 6034</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 236.05</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2426835</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 6751</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 359.44</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td rowspan="3" style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.1 μg MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 935620</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2726</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 343.21</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 687723</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1854</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 370.87</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1066234</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2148</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 496.36</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td rowspan="3" style="width:118.75pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.1 μg MAP3K7-LUC+PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 31482</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 361</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 87.16</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:15.8pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 28382</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 460</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 61.58</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.25pt"> | ||
| + | <td style="width:91.9pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 53347</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:81.95pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 562</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:80.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 94.90</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </div> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.007.png" width="313" height="311" alt="图_02" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 6. The relative luciferase activity in cells with different concentrations of transfected MAP3K7-LUC.</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold; color:#2a2b2e"> | ||
| + | 3.</span> | ||
| + | <span style="font:7pt 'Times New Roman'"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | The effect of the MAP3K7-LUC sensor as the monitor to detect the alteration of RNA splicing.</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | To test the effect of the MAP3K7-LUC sensor as the </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:24pt; text-align:justify; line-height:150%; widows:0; orphans:0; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | monitor to detect the alteration of RNA splicing, cells were transfected with plasmid sensors following treated with different concentration of PB (</span> | ||
| + | <a name="OLE_LINK7"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.6 ng/μl</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | , 0.2 ng/μl</span> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | ). The treatment with different concentration of PB represents the different degrees of RNA splicing alteration. In the presence of PB, the luminescence activity was significantly decreased in a dose dependent (Table 5 and 7, Fig 9 and 11). Moreover, based on the luciferase values in cell treated with different concentration of PB, the standard curve of MAP3K7-LUC sensor was made by EXCEL (Table 3 and 4, Fig 8 and 9). We find that the value of luciferase is dependent on the concentration of PB in cells. Based on the formula, the correlation coefficient (R</span> | ||
| + | <span style="font-family:'Times New Roman'; font-size:8pt; vertical-align:super; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | 2</span> | ||
| + | <span style="font-family:'Times New Roman'; color:#2a2b2e; background-color:#fcfdfe"> | ||
| + | value) of MAP3K7-LUC was 0.9915. </span> | ||
| + | <img src="./04_files/aratn-rw9xo.008.png" width="482" height="188" alt="修改图_03" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <a name="OLE_LINK22"> | ||
| + | </a> | ||
| + | <a name="OLE_LINK23"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 7. Cells in 24-well plates. The left is the cells before transfection and the right is the cells transfected with plasmids for 48 h.</span> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK22"> | ||
| + | </span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-indent:21pt; text-align:center; line-height:150%; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <a name="OLE_LINK4"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Table 3. The value of fluorescence in cell transfected MAP3K7-LUC with different PB treatment</span> | ||
| + | </a> | ||
| + | </p> | ||
| + | <div style="text-align:center"> | ||
| + | <table cellspacing="0" cellpadding="0" style="width:379.05pt; margin-right:auto; margin-left:auto; border-collapse:collapse"> | ||
| + | <tbody> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:116.2pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:11pt"> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK4"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc+Fluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | (Rluc+Fluc)/Rluc</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:116.2pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Blank</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 83</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 78</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1.06</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td rowspan="3" style="width:116.2pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Without PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 19107496</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 21086</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 906.16</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 30772170</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 32934</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 934.35</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.5pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 24216616</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 25174</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 961.93</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td rowspan="3" style="width:116.2pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:12pt"> | ||
| + | <a name="OLE_LINK8"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | +0.1 μl</span> | ||
| + | </a> | ||
| + | <span style="font-family:'Times New Roman'; font-size:11pt"> | ||
| + | PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 4648894</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 5868</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 792.13</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 771669</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 1207</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 638.90</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2258307</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2920</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 773.18</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td rowspan="3" style="width:116.2pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:12pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | +0.3 μl</span> | ||
| + | <span style="font-family:'Times New Roman'; font-size:11pt"> | ||
| + | PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 4256246</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 8448</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 503.81</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:16.65pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 21114296</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 42374</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 498.28</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:17.2pt"> | ||
| + | <td style="width:72.7pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 2865316</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:66.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 7968</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:79.6pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 359.56</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </div> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.009.png" width="241" height="272" alt="图_04" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 8. The relative luciferase activity in cells transfected with MAP3K7-LUC and treated with different concentration of PB.</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Table 4. </span> | ||
| + | <a name="OLE_LINK5"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | The original date of the standard curve of MAP3K7-LUC</span> | ||
| + | </a> | ||
| + | </p> | ||
| + | <div style="text-align:center"> | ||
| + | <table cellspacing="0" cellpadding="0" style="width:359.85pt; margin-right:auto; margin-left:auto; border-collapse:collapse"> | ||
| + | <tbody> | ||
| + | <tr style="height:13.6pt"> | ||
| + | <td style="width:76.25pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="-aw-bookmark-end:OLE_LINK5"> | ||
| + | </span> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:77.4pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | without PB</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:63.15pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.2 ng/μl</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:99.1pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 0.6 ng/μl</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:13.6pt"> | ||
| + | <td style="border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Luciferase</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:77.4pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 906.16</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:63.15pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 792.13</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:99.1pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 503.81</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:13.6pt"> | ||
| + | <td style="border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:77.4pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 934.35</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:63.15pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 638.90</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:99.1pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 498.28</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:13.6pt"> | ||
| + | <td style="border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | </span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:77.4pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 961.93</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:63.15pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 773.18</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:99.1pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 359.56</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr style="height:13.6pt"> | ||
| + | <td style="border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | average</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:77.4pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 934.15</span> | ||
| + | </p>oman'"> </span></p></div></body></html> | ||
| + | </td> | ||
| + | <td style="width:63.15pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 734.74</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | <td style="width:99.1pt; border-style:solid; border-width:0.75pt; padding-right:5.03pt; padding-left:5.03pt; vertical-align:middle"> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; font-size:11pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | 453.88</span> | ||
| + | </p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </div> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:justify; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'; font-weight:bold"> | ||
| + | </span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <img src="./04_files/aratn-rw9xo.010.png" width="424" height="271" alt="标曲_01" style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline"> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> | ||
| + | Fig 9. The standard curve of MAP3K7-LUC</span> | ||
| + | </p> | ||
| + | <p style="margin-top:0pt; margin-bottom:0pt; text-align:center; widows:0; orphans:0; font-size:10.5pt"> | ||
| + | <span style="font-family:'Times New Roman'"> </span></p> | ||
| + | </div> | ||
| + | </body> | ||
| + | </html> | ||
<!-- --> | <!-- --> | ||
Revision as of 15:06, 10 October 2022
The dual luciferase reporter system
According to previous studies, we found that exons of mitogen-activated protein kinase kinase kinase 7 (MAP3K7) was skipped in MDS patients. This mechanism gives rise to our idea of constructing the sensor based on the exon skipping of MAP3K7 to the plasmid containing reporter genes, which monitor the alteration of RNA splicing in cells. Our project will help us to evaluate the effectiveness of these sensors in diagnosing MDS patients in the future.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2459
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 3661