Part:BBa_K1993022

pSMA

Alpha-smooth muscle actin (α-SMA) is the actin isoform that predominates within vascular smooth-muscle cells and plays an important role in fibrogenesis.[1] Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing alpha-SMA, and their activation plays a key role in development of the fibrotic response. Previous studies have showed that α-SMA-positive hMSCs exhibited low self-renewal and lineage differentiation potential, in contrast to α-SMA-negative hMSCs, which are clonal and multi-potent. α-SMA may thus be a potential factor to determine the therapeutic potential of MSCs and the risk of MSC fibrogenesis.[2]

Tissue repair and tumor microenvironment, including inflammatory cells, neo-vasculature, and pro-fibrotic cytokines such as TGF-β, may convert MSCs into contractile myofibroblasts (MFs) that form α-smooth muscle actin (α-SMA)-containing stress fibers, which leads to fibrogenesity and reduction of the clonogenicity and adipogenic potential. Our experiment indicated the high expression of α-SMA in inflammatory condition. See results below(Figure 1, Figure 2)

Figure 1 qPCR of α-SMA in MSCs. mRNA of α-SMA was detected of high concentration in inflammatory condition, whereas in normal condition mRNA of α-SMA were not detectable.

Figure 2 In immunofluorescence, fluorescence could be observed in MSCs treated with TGF-β while not observable in control group.

As a result, it is of great importance to get the promoter of α-SMA .We acquired this gene from the plasmid we bought and purified it(Figure 3). Then we constructed it into a plasmid backbone that can express in eukaryocytes.

Figure 3 Purification of gene pSMA.

In the future, based on the promoter of α-SMA, we designed a switch under the promoter of α-SMA，which is able to detect inflammatory environment and to promote downstream genes, such as apoptosis gene(Figure 4).

Figure 4 Our future design for application of pSMA

Reference

[1]Cherng S, Young J, Ma H. Alpha-smooth muscle actin (α-SMA)[J]. J Am Sci, 2008, 4(4): 7-9.

[2] Talele N P, Fradette J, Davies J E, et al. Expression of α-smooth muscle actin determines the fate of mesenchymal stromal cells[J]. Stem cell reports, 2015, 4(6): 1016-1030.

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 1763
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 887