Part:BBa_K1993011

CXCR4-IRES-Luciferase-T2A-dtomato-T2A-hFTH

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993011 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993011 would be transducted into MSCs by lentivirus expression vector. What’s more, we confirmed its function in vitro.

Figure 1 EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A-hFTH

Details:

• Elongation factor-1α (EF-1α), a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
• CXCR4 is a chemokine receptor and its corresponding ligand is CXCL12. (Details could be seen on BBa_K1993003)
• Internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. (Details could be seen on BBa_K1993016)
• Luciferase: Firefly (Photinus pyralis) Luciferase (Details can be seen from BBa_K1993018) is a kind of oxidative enzyme that produce bioluminescence. It is able to oxidize luciferin and produce detectable bioluminescence. It’s convenient to observe biological processes, especially allowing for non-invasive observation of cells.
• T2A： Between every two protein coding sequence, we added T2A, a good candidate to replace IRES because of its small size and high cleavage efficiency between every two protein coding sequences to ensure all the coding sequences express as expected. (Details can be seen from BBa_K1993019)
• dTomato (Details can be seen from BBa_K1993020) is a kind of red fluorescent protein used for observation in vitro conveniently and easily;
• hFTH (See details in BBa_K1993021), is another protein that could be observed in vivo by MRI.

Results:

Figure 1. Expression of dTomato in 293FT cells.

Figure 2. Expression of dTomato in MSCs.

Future work:

Since it is unreasonable to apply dTomato and luciferase to human bodies, we improved our device with hFTH. Making use of functional imaging, we would overexpress hFTH to be reported by MRI in vivo. As a matter of time, we didn’t confirm the function of this part in most in vivo and in vitro, but we decided to apply them to preclinical and clinical pharmaceutical research in the future.

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 494
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2496