Difference between revisions of "Part:BBa K1431302"
 
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Generating stable lines in cells expressing the SV40 large T antigen is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. This is because the SV40 large T antigen induces DNA replication at the SV40 ori in the vector sequence. If this occurs after the vector has integrated into the genome, it will result in isolated regions of the chromosome trying to replicate  
 
Generating stable lines in cells expressing the SV40 large T antigen is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. This is because the SV40 large T antigen induces DNA replication at the SV40 ori in the vector sequence. If this occurs after the vector has integrated into the genome, it will result in isolated regions of the chromosome trying to replicate  
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illegitimately, leading to chromosomal duplications and generally messing up the cell's genome. The best recommendation would be to use a cell line that does not express the SV40 large T antigen. Deleting the origin could be an option, (50), however, this may affect promoter activity. Transient transfection of SV40 ori-containing plasmids into cells expressing the SV40 large T antigen results in replication of the plasmid (without hurting the host cell's chromosomes) and therefore an amplification of the gene of interest and subsequently very high levels of expression. Hence, we recommend the SV40 ori-containing vectors for transient use only in SV40 large T antigen-containing cells, but they can be used both transiently and stably in cells that do not have the SV40 large T antigen.(Source:lifetechnoligies)
+
illegitimately, leading to chromosomal duplications and generally messing up the cell's genome. The best recommendation would be to use a cell line that does not express the SV40 large T antigen. Deleting the origin could be an option, however, this may affect promoter activity. Transient transfection of SV40 ori-containing plasmids into cells expressing the SV40 large T antigen results in replication of the plasmid (without hurting the host cell's chromosomes) and therefore an amplification of the gene of interest and subsequently very high levels of expression. Hence, we recommend the SV40 ori-containing vectors for transient use only in SV40 large T antigen-containing cells, but they can be used both transiently and stably in cells that do not have the SV40 large T antigen.(Source:[http://tools.lifetechnologies.com/content/sfs/productnotes/F_071215_MammalianExpressionVectors.pdf lifetechnoligies])
  
  

Latest revision as of 03:44, 18 October 2014

SV40 promoter+SV40 PolyA

The SV40 (Simian Virus 40) promoter contains the SV40 enhancer promoter region and origin of replication for high-level expression and replication in cell lines expressing the large T antigen. It contains two 72bp SV40 enhancer repeats.

Mammalian vectors that contain the SV40 polyoma ori (usually part of the SV40 early promoter in our vectors) replicate episomally, but only in cells that express the SV40 large T antigen (e.g. COS-7 and 293T cells). They will not replicate episomally in the absence of the SV40 large T antigen.

Generating stable lines in cells expressing the SV40 large T antigen is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. This is because the SV40 large T antigen induces DNA replication at the SV40 ori in the vector sequence. If this occurs after the vector has integrated into the genome, it will result in isolated regions of the chromosome trying to replicate illegitimately, leading to chromosomal duplications and generally messing up the cell's genome. The best recommendation would be to use a cell line that does not express the SV40 large T antigen. Deleting the origin could be an option, however, this may affect promoter activity. Transient transfection of SV40 ori-containing plasmids into cells expressing the SV40 large T antigen results in replication of the plasmid (without hurting the host cell's chromosomes) and therefore an amplification of the gene of interest and subsequently very high levels of expression. Hence, we recommend the SV40 ori-containing vectors for transient use only in SV40 large T antigen-containing cells, but they can be used both transiently and stably in cells that do not have the SV40 large T antigen.(Source:[http://tools.lifetechnologies.com/content/sfs/productnotes/F_071215_MammalianExpressionVectors.pdf lifetechnoligies])


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]