Determination of Intracellular Condensates Based on Protein Knockout

Liquid-liquid phase separation (LLPS) is a central player in the assembly of membrane-free compartments for biomolecular condensates. Various proteins, RNA and other biomolecules undergo LLPS and exhibit a variety of cellular functions. Researchers need to carefully design experiments to manipulate the phase separation of proteins without altering other functions or properties of biomolecular condensates. Guided by sequence analysis, mutations can be introduced into proteins to alter their phase separation properties. However, mutations of phase-separated proteins may not be as straightforward as for structural proteins. For example, altering the phase behavior of low complexity proteins. Developments in genetic engineering have played a very important role in the field of studying LLPS-controlled protein processes.

Fig. 1. A model of Tet1-mediated chromocenter clustering in DNA hypomethylated cells. (Hagihara Y, et al., 2021)

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In addition to providing reliable experimental tools to assay LLPS in vitro, our experts are dedicated to extracting information about functional phase separation from the large number of natural sequence variants available in the post-genomic era, aiming to develop innovative tools to read this information and identify the key features and selection pressures behind phase separation in vivo. Here, CD BioSciences offers knockout and gene silencing methods to analyze LLPS-controlled protein processes in vivo.

These approaches have the advantage of allowing the analysis of each protein in a biomolecular condensed object to separate proteins that affect the formation of membraneless organelles (MLOs) from those that do not affect MLO biogenesis. The main strategies are as follows:

• Knockout/knockdown of a gene encoding a protein of interest with a predisposition to LLPS
  Sarcoma fusion protein (FUS) is a prion-like RNA-binding protein and is a typical example of an intrinsically disordered protein. We can analyze how LLPS controls phase behavior by knocking out the endogenous FUS gene.
• Knockout/knockdown of partners of this target squirrel
  We can knockout cell lines of endogenous proteins of interest and perform subsequent exogenous expression of their individual structural domains to investigate the propensity of tumor regions to phase segregate. This approach makes it possible to isolate the kinesins involved in MLO initiation.

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• The phase behavior of individual proteins can be characterized.
• The saturation concentration, stimulus responsiveness and material properties of the cohesive phase can be predicted from the sequence.
• Once variants with specific phase separation defects have been identified, they can be rapidly introduced into cells to replace wild-type proteins.
• The resulting cell lines can be tested for their ability to promote membrane-free compartment formation under physiological conditions or in response to perturbations.

Both proteins and RNAs can be used as partners. We offer cutting-edge genetic engineering strategies to analyze LLPS-controlled protein processes in vivo. In addition, we attempt to develop RNA silencing assays to study the role of RNA. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.