Difference between revisions of "Part:BBa K1993008"

Revision as of 12:31, 17 October 2016

Luciferase-IRES-eGFP

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993008 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993008 would be transduced into MSCs by lentivirus expression vector. What’s more, we confirmed its function in vitro and in vivo (DTH).

Figure 1 EF-1α-CXCR5-IRES-eGFP

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.
• CXCR5 is a chemokine receptor and its corresponding ligand is CXCL13. (Details could be seen on BBa_K1993013)
• 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)
• LeGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of GFPs 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 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 CXCL13. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs had improved.

In vivo

1. 
   
   Figure8
   Figure8 :The inflamed ears were collected from each group and subjected to in situ immunofluorescence staining. Our results revealed that MSC^CXCR5 exhibited enhanced capacities for targeted migration to the ears in DTH model.
2. 
   
   Figure9
   The inflamed ears were collected from each group, subjected to in situ immunofluorescence staining and observed under fluorescence microscope. Our results revealed MSC^CXCR5 could be located in the inflamed ears of DTH mice.
   
3. 
   
   Figure 10
   Ear sampling for quantitative PCR analysis. Compared with DTH+MSCs group, levels of anti-inflammatory cytokines (IL-10) elevated in DTH+MSC^CXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSC^CXCR5 group. In a word, MSC^CXCR5 displayed greater immunoregulatory effect.
4. 
   
   Figure 11
   
   Figure 12
   After injection, MSC^CXCR5 displayed better treatment efficacy than MSCeGFP, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSC^CXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection. In a word, MSC^CXCR5 regulated immune system in inflammatory condition in a more significant way.
   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 228
     Illegal BamHI site found at 771
     
   • 23
     COMPATIBLE WITH RFC[23]
   • 25
     COMPATIBLE WITH RFC[25]
   • 1000
     COMPATIBLE WITH RFC[1000]