Part:BBa_K1993009

CXCR4-T2A-Luciferase-IRES-eGFP

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993009 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993009 would be transduced into MSCs by lentivirus expression vector. What’s more, we would confirm its function in vitro and in vivo (inflammatory bowel disease animal models, IBD).

Figure 1 EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP

Details:

• Elongation factor-1α (EF-1α), as 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)
• T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. (Details could be seen on BBa_K1993019)
• Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. (Details could be seen on BBa_K1993015)
• 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)
• eGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of GFP in mammalian cells. (Details could be seen on BBa_K1993017)

Results:

In vitro

1. Figure 2	Figure 3

   mRNA and protein levels of CXCR4 of MSCs and our modified MSCs were semi-quantified and detected by qPCR and western blot, respectively. mRNA (Figure 2) and protein levels (Figure 3) of CXCR4 both elevated in our modified MSCs.

2. Figure 4	Figure 5

   As for expression of eGFP, transfected 293FT cells (Figure 4) and MSCs (Figure 5) were observed under fluorescent microscope. As a result, eGFP was expressed.

3. Figure 6	Figure 7

   Chemotaxis of engineered MSCs were examined against CXCL12. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs improved.

In vivo

1. 
   
   Figure8 :Under IVIS Spectrum, MSCs^CXCR4 exhibited enhanced capacities for targeted migration to the bowels in IBD model.
2. 
   
   Figure 9:Under IVIS Spectrum, MSCs^CXCR4 could be detected and located in IBD mice.
   
   
3. 
   

   Figure 10	Figure 11
   Figure 12	Figure 13

   Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSC^CXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSC^CXCR4 group.
4. 
   

   Figure 14	Figure 15
   Figure 16	Figure 17

   After injecting with MSCs^CXCR4, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.
5. 
   
   Figure 18 Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCs^CXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.
   
   In a word, MSCs^CXCR4 have greater immunoregulatory function.
   
   
   Sequence and Features

   Assembly Compatibility:

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

     Illegal BglII site found at 1350
     Illegal BamHI site found at 494
     Illegal BamHI site found at 1893
     
   • 23
     COMPATIBLE WITH RFC[23]
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     COMPATIBLE WITH RFC[25]
   • 1000
     COMPATIBLE WITH RFC[1000]