schliessen

Filtern

 

Bibliotheken

Construction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes

The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1] . But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has... Full description

Journal Title: PLoS ONE 2013, Vol.8(4)
Main Author: Bai, Jiasi
Other Authors: Li, Jungang , Mao, Qing
Format: Electronic Article Electronic Article
Language:
Subjects:
ID: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0061412 ; PMCID: 3634076 ; PMID: 23626683
Zum Text:
SendSend as email Add to Book BagAdd to Book Bag
Staff View
recordid: pubmed_central3634076
title: Construction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes
format: Article
creator:
  • Bai, Jiasi
  • Li, Jungang
  • Mao, Qing
subjects:
  • Research Article
  • Medicine
ispartof: PLoS ONE, 2013, Vol.8(4)
description: The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1] . But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2] . In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.
language:
source:
identifier: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0061412 ; PMCID: 3634076 ; PMID: 23626683
fulltext: fulltext
issn:
  • 1932-6203
  • 19326203
url: Link


@attributes
ID1382221457
RANK0.07
NO1
SEARCH_ENGINEprimo_central_multiple_fe
SEARCH_ENGINE_TYPEPrimo Central Search Engine
LOCALfalse
PrimoNMBib
record
control
sourcerecordid3634076
sourceidpubmed_central
recordidTN_pubmed_central3634076
sourceformatXML
sourcesystemPC
pqid1346595013
galeid478152431
display
typearticle
titleConstruction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes
creatorBai, Jiasi ; Li, Jungang ; Mao, Qing
contributorWu, Yuntao (editor)
ispartofPLoS ONE, 2013, Vol.8(4)
identifier
subjectResearch Article ; Medicine
descriptionThe SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1] . But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2] . In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.
source
version9
lds50peer_reviewed
links
openurl$$Topenurl_article
openurlfulltext$$Topenurlfull_article
search
creatorcontrib
0Bai, Jiasi
1Li, Jungang
2Mao, Qing
3Wu, Yuntao
titleConstruction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes
descriptionThe SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1] . But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2] . In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.
subject
0Research Article
1Medicine
general
023626683
13634076
210.1371/journal.pone.0061412
3PMC (PubMed Central)
sourceidpubmed_central
recordidpubmed_central3634076
issn
01932-6203
119326203
rsrctypearticle
creationdate2013
addtitlePLoS ONE
searchscopepubmed_central
scopepubmed_central
lsr30VSR-Enriched:[galeid, pqid, pages]
sort
titleConstruction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes
authorBai, Jiasi ; Li, Jungang ; Mao, Qing
creationdate20130423
facets
frbrgroupid8985026809535592224
frbrtype5
creationdate2013
topic
0Research Article
1Medicine
collectionPMC (PubMed Central)
prefilterarticles
rsrctypearticles
creatorcontrib
0Bai, Jiasi
1Li, Jungang
2Mao, Qing
3Wu, Yuntao
jtitlePlos One
toplevelpeer_reviewed
delivery
delcategoryRemote Search Resource
fulltextfulltext
addata
aulast
0Bai
1Li
2Mao
3Wu
aufirst
0Jiasi
1Jungang
2Qing
3Yuntao
au
0Bai, Jiasi
1Li, Jungang
2Mao, Qing
3Wu, Yuntao
atitleConstruction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes
jtitlePLoS ONE
risdate20130423
volume8
issue4
eissn1932-6203
formatjournal
genrearticle
ristypeJOUR
abstractThe SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1] . But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2] . In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.
pubPublic Library of Science
doi10.1371/journal.pone.0061412
pmid23626683
pagese61412
oafree_for_read
date2013-04-23