Part:BBa_K1993005

CXCR5-IRES-eGFP

In consideration of testifying the location of MSCs in multiple ways and provide future teams with optional and economic way to observe, we enhanced the basic part of RLuc with the gene of eGFP and improved a previous part (For more details, see [1]) .

eGFP is a type of GFP derivatives by mutation. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. eGFP is one of the most used fluorescent proteins which is easy to detect. In our experiments, we applied eGFP in order to report the expression of Luciferase and to show the actual sites of MSCs in vitro.   Another marking protein we engineered to MSCs is Luciferase. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence by oxidizing luciferin substrate and responsible for regulating light production in a variety of organs. It’s convenient to observe biological processes, especially allowing for observation of cells non-invasively and specifically. In this composite part, we chose the gene of Renilla-luciferin 2-monooxygenase (RLuc) (936 bp). Unlike Firefly Luciferase (Fluc) requiring ATP to catalyze, RLuc is a shorter version cooperating with O2 to oxidize luciferin. （Details can be seen from [parts.igem.org/Part:BBa_K1993018 BBa_K1993018]) When MSCs engineered with coding sequence of Luciferase were administrated to mice that were injected intraperitoneally with Luciferin simultaneously, those areas accumulated with higher concentration of MSCs would oxidize more luciferin therefore display stronger fluorescence in IVIS spectrum system.

On one hand, the advantages of eGFP lie in convenience as well as low cost for observation comparing to Luciferase. On the other hand, Luciferase make up for the shortage of eGFP as oxidized Luciferin could be observed in living animals， which expands its application range and lays a foundation for its further clinical application. The observation of these two complementary marking proteins ensures accurate locating of MSCs both in vitro and in vivo.

To ensure co-expression of Luciferase and eGFP, we introduced internal ribosome entry site (IRES) between them, an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex. The location for IRES elements is often in the 5'UTR, but can also occur elsewhere in mRNAs.   IRES has been widely used due to the following advantages: (1) ensured co-expression of genes before and after the IRES; (2) feasibility of adding subcellular localization sequences to the gene after IRES; and (3) availability of commercial expression plasmids harboring IRES. [2]   In our project, firstly we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Then we found the fantastic function of IRES and purified it (Figure 1). Taking advantage of function of IRES, we constructed a plasmid that IRES linked between two protein coding genes. For example, we constructed BBa_K1993005 (Luciferase-IRES-eGFP) to ensure eGFP could be expressed.

In our experiments, we confirmed its expression by fluorescent microscope and IVIS spectrum system. The results are shown as follows:

Figure1

We first testified the expression of eGFP in 293FT cells. As a result, eGFP had been successfully transduced into 293FT cells. (Figure 1)

Figure2.

We transfected MSCs with our composite part. Figure 2 indicates successful expression of eGFP in MSCs.

Figure3.

After intraperitoneal injection of Luciferase, MSCs engineered with Luciferase and eGFP were applied to mouse. Figure 3 showed the signal of oxidized Luciferin in living mice.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 31
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 973
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 259
  Illegal SapI site found at 1050