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 (IBD).

Figure 1 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) <lT2A 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 GFPs in mammalian cells. (Details could be seen on [htttp://parts.igem.org/Part:BBa_K1993017 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 293T cells (Figure 4) and MSCs (Figure 5) were observed under fluorescent microscope. As a result, eGFP were 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, MSCsCXCR4 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.
   
   In a word, MSCs^CXCR4 have greater immunoregulatory function.
4. 
   

   Figure 10	Figure 11
   Figure 12	Figure 13

   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. In a word, MSCs^CXCR4have greater immunoregulatory function.
5. 
   
   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:

   • 10
     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]
   • 25
     COMPATIBLE WITH RFC[25]
   • 1000
     COMPATIBLE WITH RFC[1000]