Part:BBa_K404090

[AAV2]-Rep40ex

Usage and Biology

beta-globin intron
BioBrick Nr.	BBa_K404107
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_MCS
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Providing an element assumed to be an enhancer of transgene expression (Nott, Meislin, & Moore, 2003), the iGEM team Freiburg presents a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest.
Introns are non-coding sequences that are spliced after gene transcription. Apart from the possibility of alternative splicing which leads to an increased variability of translated proteins from one single gene, additional functions of introns have been found. They regulate and enhance gene expression at multiple levels such as initiating transcription, gene editing and polyadenylation of pre-mRNA (Nott, Le Hir, & Moore, 2004).In Valencia, Dias, & Reed (2008) the authors described the influence of splicing transgenes containing one intron at the 5´ and 3´ position, respectively. They demonstrated that splicing promotes the nuclear export of mRNA and that the spliceosome is cross-coupled to the mRNA export machinery.

Characterization

The BioBrick part beta-globin intron consists partially of a chimeric CMV promoter, followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 20 nucleotides of exon 3 of the beta globin gene. Our BioBrick part beta globin intron is assumed to enhance eukaryotic gene expression.
AAV-293 cells were transfected with all genes necessary for producing viral particles encapsidating two different rAAV genomes with and without beta-globin intron respectively. mVenus expression was determined by flow cytometry 24-hours post infection of HT1080 cells. The rAAV genomes missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results, we suggest using the beta-globin intron in dependence on the size of your transgene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 53
  Illegal XhoI site found at 920
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]