Part:BBa_K1993029

CXCR4-T2A-Luciferase-IRES-eGFP

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993029 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993029 would be transduced into MSCs by lentivirus expression vectors. 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

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.
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.
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

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


Figure 2	Figure 3


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Figure 10	Figure 11

Figure 12	Figure 13