Difference between revisions of "Part:BBa K4839024"
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<p>Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.</p>
 
<p>Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.</p>
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 +
<p>[1] Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006).</p>
 +
<p>[2] Shin, Y. J. et al. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep 5, 14269 (2015).</p>
 +
<p>[3] Shen, H. et al. MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension. Circulation 142, 1190–1204 (2020).</p>
 +
<p>[4] Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947–953 (2020).</p>
  
  

Revision as of 17:22, 3 October 2023


pGK-GFP-IRF4

This part is design for the validification of our BioPROTC system. In our design, we fused GFP and IRF4 together and thus we can use both anti-GFP or anti-IRF4 BioPROTAC to detect the degradation efficiency of both protein.

we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 3-2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.


Figure1. Western blot signing degrdation of IRF4. "D" represents doxycycline.

Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.

[1] Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3, 887–889 (2006).

[2] Shin, Y. J. et al. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep 5, 14269 (2015).

[3] Shen, H. et al. MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension. Circulation 142, 1190–1204 (2020).

[4] Klichinsky, M. et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 38, 947–953 (2020).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 257
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2527
    Illegal SpeI site found at 257
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2627
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 257
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 257
    Illegal NgoMIV site found at 2052
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 99
    Illegal BsaI.rc site found at 2358
    Illegal SapI site found at 1510
    Illegal SapI.rc site found at 1441