Part:BBa_K2889003

pSB1C3-miR-21-sponge-4s

Our previous project constructed a mir-21 sponge containing marker gene of GFP for the detection and inhibition mir-21 in the cell lines, and offers a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer in the future. miRNA sponge, which contains multiple target sites complementary to a miRNA of interest, is used to be a inhibitor of miRNA. We proposed miRNA sponge could be a monitor to reflect the change of miRNA expression in cells. In our previous project, we constructed two miR-21 sponge plasmid, pEGFP-miR-21-sponge-2s and pEGFP-miR-21-sponge-6s, as the monitor to detect the expression of miR-21 in cells. We hoped to improved the sensitive of miR-21-sponge as a monitor. We designed miR-21 sponges contains four miR-21 binding sites with 3-nt spacers for bulged sites based on the sequence of hsa-miR-21. To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-4s with different concentration of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula listed below.

    copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)

The standard curve of miR-21-sponge-4s were made by EXCEL. We find that when miR-21 copies changed the slope of the standard curve of miR-21-sponge-4s was greater than miR-21-sponge-2s and miR-21-sponge-6s which indi-cated miR-21-sponge-4s is more sensitive than miR-21-sponge-2s and miR-21-sponge-6s.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 66
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]