Difference between revisions of "Part:BBa K3335003"
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===KRAS siRNA overexpressed in HEK293T cell and exosome===
 
===KRAS siRNA overexpressed in HEK293T cell and exosome===
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<p>First, we detected the correct expression of KRAS siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siRK is this part. CMV-iRGD-siR<sup>P+C+K</sup> is a Composite part.</p>
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<p>First, we detected the correct expression of KRAS siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siR<sup>K</sup> is this part. CMV-iRGD-siR<sup>P+C+K</sup> is a Composite part.</p>
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https://2020.igem.org/wiki/images/f/f4/T--NJU-China--2.png
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<p>Figure.1 KRAS siRNA is overexpressed is HEK293T cell</p>
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https://2020.igem.org/wiki/images/8/8e/T--NJU-China--3.png
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<p>Figure2. KRAS siRNA is overexpressed in exosome</p>
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https://2020.igem.org/wiki/images/4/42/T--NJU-China--4.png
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<p>Figure.3 Absolute quantification of KRAS siRNA</p>
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<p>At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).</p>
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<p>According to the absolute quantification of KRAS siRNA, we found that the amount of siRNA in HEK293T cells is 4.569E-06 nmol/L, the amount of siRNA in exosome is 2.848E-07 nmol/L. The exosome siRNA is equivalent to 6% of the siRNA concentration in the cell.</p>

Revision as of 11:44, 22 October 2020

We used it to express iRGD-Lamp2b and siRNA targeted at KRAS

We used it to express iRGD-Lamp2b and siRNA targeted at KRAS Lung cancer is the major cause of cancer-related deaths globally. Mutant KRAS is a feature of 15%–50% of lung cancer cases[1]. Unfortunately, although much effort has been spent on searching for small molecule inhibitors of KRAS, KRAS gene has proven extraordinarily difficult to target by current pharmacological agents.

This year we developed an alternative strategy to silence the so-called untargetable and undruggable KRAS gene by employing exosome-mediated siRNA delivery.At the same time, we used iRGDLamp2b to achieve exosome targeting. Particularly, we reprogrammed cells to simultaneously express KRAS siRNA and Lamp2b. Lamp2b is an exosomal membrane protein, in fusion with a tumor-penetrating internalizing RGD (iRGD) peptide (CRGDKGPDC), and then produce the tumor-targeting exosomes as KRAS siRNA delivery system.[1]

With this system, we can target tumor cells and deliver siRNA.

[1]Yu Zhou et al.,Tumor-specific delivery of KRAS siRNA with iRGD-exosomes efficiently inhibits tumor growth. ExRNA,2019.

Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

KRAS siRNA overexpressed in HEK293T cell and exosome

First, we detected the correct expression of KRAS siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siRK is this part. CMV-iRGD-siRP+C+K is a Composite part.

T--NJU-China--2.png

Figure.1 KRAS siRNA is overexpressed is HEK293T cell

T--NJU-China--3.png

Figure2. KRAS siRNA is overexpressed in exosome

T--NJU-China--4.png

Figure.3 Absolute quantification of KRAS siRNA

At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

According to the absolute quantification of KRAS siRNA, we found that the amount of siRNA in HEK293T cells is 4.569E-06 nmol/L, the amount of siRNA in exosome is 2.848E-07 nmol/L. The exosome siRNA is equivalent to 6% of the siRNA concentration in the cell.