Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes
A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the rel...
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| Veröffentlicht in: | Pharmaceutical research 2002-04, Vol.19 (4), p.377-381 |
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| creator | TACHIBANA, Rieko HARASHIMA, Hideyoshi IDE, Naoko UKITSU, Sachiko OHTA, Yasuko SUZUKI, Norio KIKUCHI, Hiroshi SHINOHARA, Yasuo KIWADA, Hiroshi |
| description | A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed.
AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification.
Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose.
These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors. |
| doi_str_mv | 10.1023/A:1015162722295 |
| format | Article |
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AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification.
Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose.
These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.</description><identifier>ISSN: 0724-8741</identifier><identifier>EISSN: 1573-904X</identifier><identifier>DOI: 10.1023/A:1015162722295</identifier><identifier>PMID: 12033367</identifier><identifier>CODEN: PHREEB</identifier><language>eng</language><publisher>New York, NY: Springer</publisher><subject>Animals ; Biological and medical sciences ; Cations ; Cell Nucleus - drug effects ; Cell Nucleus - genetics ; Cell Nucleus - metabolism ; Dose-Response Relationship, Drug ; Drug Delivery Systems - methods ; Drug Evaluation, Preclinical - methods ; Efficiency ; Gene expression ; Gene Expression Regulation - drug effects ; Gene Expression Regulation - physiology ; General pharmacology ; Genetic testing ; Liposomes ; Medical sciences ; Pharmaceutical technology. Pharmaceutical industry ; Pharmacology. Drug treatments ; Plasmids ; Plasmids - administration & dosage ; Plasmids - genetics ; Polymerase Chain Reaction - methods ; Rats ; Transfection - methods ; Tumor Cells, Cultured ; Vectors (Biology)</subject><ispartof>Pharmaceutical research, 2002-04, Vol.19 (4), p.377-381</ispartof><rights>2002 INIST-CNRS</rights><rights>Copyright Kluwer Academic Publishers Apr 2002</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c356t-4ef9831b48a7706f1885c76e3aa481e6b09443e766a6ddb890ea2a390e103fd03</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=13701556$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12033367$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>TACHIBANA, Rieko</creatorcontrib><creatorcontrib>HARASHIMA, Hideyoshi</creatorcontrib><creatorcontrib>IDE, Naoko</creatorcontrib><creatorcontrib>UKITSU, Sachiko</creatorcontrib><creatorcontrib>OHTA, Yasuko</creatorcontrib><creatorcontrib>SUZUKI, Norio</creatorcontrib><creatorcontrib>KIKUCHI, Hiroshi</creatorcontrib><creatorcontrib>SHINOHARA, Yasuo</creatorcontrib><creatorcontrib>KIWADA, Hiroshi</creatorcontrib><title>Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes</title><title>Pharmaceutical research</title><addtitle>Pharm Res</addtitle><description>A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed.
AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification.
Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose.
These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cations</subject><subject>Cell Nucleus - drug effects</subject><subject>Cell Nucleus - genetics</subject><subject>Cell Nucleus - metabolism</subject><subject>Dose-Response Relationship, Drug</subject><subject>Drug Delivery Systems - methods</subject><subject>Drug Evaluation, Preclinical - methods</subject><subject>Efficiency</subject><subject>Gene expression</subject><subject>Gene Expression Regulation - drug effects</subject><subject>Gene Expression Regulation - physiology</subject><subject>General pharmacology</subject><subject>Genetic testing</subject><subject>Liposomes</subject><subject>Medical sciences</subject><subject>Pharmaceutical technology. Pharmaceutical industry</subject><subject>Pharmacology. Drug treatments</subject><subject>Plasmids</subject><subject>Plasmids - administration & dosage</subject><subject>Plasmids - genetics</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Rats</subject><subject>Transfection - methods</subject><subject>Tumor Cells, Cultured</subject><subject>Vectors (Biology)</subject><issn>0724-8741</issn><issn>1573-904X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNpdkU1rFTEUhoMo9lpdu5Mg6G5qvibJuCulVaEggoK74UzmjKZkMmOSsb2_wr9sbr0iuDpwzvO-i-cQ8pyzM86EfHP-ljPeci2MEKJrH5Adb41sOqa-PiQ7ZoRqrFH8hDzJ-YYxZnmnHpMTLpiUUpsd-fVpg1h8geJ_IoUIYZ99pstE3ZIShrpfIh2w3CJGGrd5wHS4xs0FhETXAHn2Y67RkX7DiBTv1oQ5H2LgaqsvewpTqbGSIOYJ3X3lrS_fqbuv944Gvy55mTE_JY8mCBmfHecp-XJ1-fnifXP98d2Hi_PrxslWl0bh1FnJB2XBGKYnbm3rjEYJoCxHPbBOKYlGa9DjONiOIQiQdXAmp5HJU_L6T--alh8b5tLPPjsMASIuW-4Nr-6sFhV8-R94s2ypesp9Va6tFPIAvThC2zDj2K_Jz5D2_V_PFXh1BCA7CFM14Xz-x0lT39hq-Rs7io9N</recordid><startdate>20020401</startdate><enddate>20020401</enddate><creator>TACHIBANA, Rieko</creator><creator>HARASHIMA, Hideyoshi</creator><creator>IDE, Naoko</creator><creator>UKITSU, Sachiko</creator><creator>OHTA, Yasuko</creator><creator>SUZUKI, Norio</creator><creator>KIKUCHI, Hiroshi</creator><creator>SHINOHARA, Yasuo</creator><creator>KIWADA, Hiroshi</creator><general>Springer</general><general>Springer Nature B.V</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7RV</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>KB0</scope><scope>M0S</scope><scope>M1P</scope><scope>NAPCQ</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope></search><sort><creationdate>20020401</creationdate><title>Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes</title><author>TACHIBANA, Rieko ; HARASHIMA, Hideyoshi ; IDE, Naoko ; UKITSU, Sachiko ; OHTA, Yasuko ; SUZUKI, Norio ; KIKUCHI, Hiroshi ; SHINOHARA, Yasuo ; KIWADA, Hiroshi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c356t-4ef9831b48a7706f1885c76e3aa481e6b09443e766a6ddb890ea2a390e103fd03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cations</topic><topic>Cell Nucleus - drug effects</topic><topic>Cell Nucleus - genetics</topic><topic>Cell Nucleus - metabolism</topic><topic>Dose-Response Relationship, Drug</topic><topic>Drug Delivery Systems - methods</topic><topic>Drug Evaluation, Preclinical - methods</topic><topic>Efficiency</topic><topic>Gene expression</topic><topic>Gene Expression Regulation - drug effects</topic><topic>Gene Expression Regulation - physiology</topic><topic>General pharmacology</topic><topic>Genetic testing</topic><topic>Liposomes</topic><topic>Medical sciences</topic><topic>Pharmaceutical technology. Pharmaceutical industry</topic><topic>Pharmacology. Drug treatments</topic><topic>Plasmids</topic><topic>Plasmids - administration & dosage</topic><topic>Plasmids - genetics</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Rats</topic><topic>Transfection - methods</topic><topic>Tumor Cells, Cultured</topic><topic>Vectors (Biology)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>TACHIBANA, Rieko</creatorcontrib><creatorcontrib>HARASHIMA, Hideyoshi</creatorcontrib><creatorcontrib>IDE, Naoko</creatorcontrib><creatorcontrib>UKITSU, Sachiko</creatorcontrib><creatorcontrib>OHTA, Yasuko</creatorcontrib><creatorcontrib>SUZUKI, Norio</creatorcontrib><creatorcontrib>KIKUCHI, Hiroshi</creatorcontrib><creatorcontrib>SHINOHARA, Yasuo</creatorcontrib><creatorcontrib>KIWADA, Hiroshi</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>ProQuest Central (Corporate)</collection><collection>Nursing & Allied Health Database</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Nursing & Allied Health Premium</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><jtitle>Pharmaceutical research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>TACHIBANA, Rieko</au><au>HARASHIMA, Hideyoshi</au><au>IDE, Naoko</au><au>UKITSU, Sachiko</au><au>OHTA, Yasuko</au><au>SUZUKI, Norio</au><au>KIKUCHI, Hiroshi</au><au>SHINOHARA, Yasuo</au><au>KIWADA, Hiroshi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes</atitle><jtitle>Pharmaceutical research</jtitle><addtitle>Pharm Res</addtitle><date>2002-04-01</date><risdate>2002</risdate><volume>19</volume><issue>4</issue><spage>377</spage><epage>381</epage><pages>377-381</pages><issn>0724-8741</issn><eissn>1573-904X</eissn><coden>PHREEB</coden><abstract>A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed.
AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification.
Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose.
These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.</abstract><cop>New York, NY</cop><pub>Springer</pub><pmid>12033367</pmid><doi>10.1023/A:1015162722295</doi><tpages>5</tpages></addata></record> |
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| identifier | ISSN: 0724-8741 |
| ispartof | Pharmaceutical research, 2002-04, Vol.19 (4), p.377-381 |
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| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | Animals Biological and medical sciences Cations Cell Nucleus - drug effects Cell Nucleus - genetics Cell Nucleus - metabolism Dose-Response Relationship, Drug Drug Delivery Systems - methods Drug Evaluation, Preclinical - methods Efficiency Gene expression Gene Expression Regulation - drug effects Gene Expression Regulation - physiology General pharmacology Genetic testing Liposomes Medical sciences Pharmaceutical technology. Pharmaceutical industry Pharmacology. Drug treatments Plasmids Plasmids - administration & dosage Plasmids - genetics Polymerase Chain Reaction - methods Rats Transfection - methods Tumor Cells, Cultured Vectors (Biology) |
| title | Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes |
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