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How to characterize CRISPR/CAS9 RANKL knockout mesenchymal stem cell clones


Are you curious about how you can make single-cell dispensing easier? 


Then you have come to the right spot. 

In this article, we will cover how researchers use the Cellink F.SIGHT to ease their workflow. 

Investing in new technology is alluring because you know what you stand to win— easier workflows. But there’s a real risk that your efforts won’t pay off - and you’ll lose precious time spent on a fruitless attempt. 

Knowing how to optimize your work and finding where to start can feel overwhelming because of the many moving parts that are involved. 

If you're wondering how you can make single-cell dispensing easier, you have come to the right place. 
 

Genetically engineered single-cell clones

 
Pharmaceutical firms all over the world see the characterization of CRISPR/Cas9-engineered cells as a top priority. The same is true for novel cellular therapeutic applications and regenerative medical uses.  

That is why the team at the Department of Microsystems Engineering at the University of Freiburg refined a modern workflow to improve this. 

This state-of-the-art, profitable and smooth-running workflow incorporates the Cellink F.SIGHT fluorescent single-cell dispensing technology, a genomic editing screen called surveyor assay, RT-PCR mRNA assessing altered gene expression, and a versatile protein detection tool called emulsion coupling.

Laboratory workflow
 

Improve the identification and characterization CRISPR/CAS9 knockout technology

 
As part of their experimental proof of concept, the team genetically modified immortalized mesenchymal stem cells (MSCs). They did this to enhance their ability to form bone for possible regenerative medical applications. 

They deleted The Tumor Necrosis Factor Superfamily Member 11 (TNFSF11). This encodes the nuclear factor kappa B ligand activating receptor (RANKL). 

RANKL has formerly played a central role in bone homeostasis. This is due to it orchestrating the balance between osteoblasts that generate bone - and osteoclasts that degrade it. 

This is mainly because RANKL is expressed in bone tissue by mesenchymal stem cells, osteoblasts and T cells. When RANKL is present, the nuclear factor kappa B activating receptor (RANK) activates. 

This stimulates pre-osteoclasts to differentiate into osteoclasts. This degrade bone, and for bone formation, MSCs differentiate into osteoblasts and deposit calcified structures.  

Genetically modified MSCs and their progenitors can no longer recruit osteoclasts by knocking out TNFSF11. For this reason, researchers can improve bone formation on the sidelines of their implantation in a regenerative medical application. 

The instrument used

 
One of the technologies that most aided the development of this workflow is Cellink's single-cell isolation technology. 

This is an easy-to-use, affordable and highly controlled technology. It is also applicable for a wide range of specific cell cloning applications ranging from 0.8 μm prokaryotes to 100 μm plant cells. Single-cell dispensing is based on inkjet-type technology. 

This generates free-flying microdroplets on demand that encapsulate cells. It deposits them on variable substrates using a non-contact delivery process. 

The team chose the Cellink F.SIGHT single-cell dispenser to develop the workflow. This was because of its benefits over methods like fluorescence-associated cell sorting (FACS).

While researchers use FACS for single-cell isolation, that sorting process has a negative effect on the cloning efficiency of susceptible or partially damaged cells.

This is because it applies high shear forces and electrostatic charge to the cells.

F.SIGHT

How the F.SIGHT works

 
The F.SIGHT is partly applicable for cloning of engineered cells. In addition to preserving their viability, it provides direct evidence of single-cell clonality. It also has both native and fluorescent sorting capabilities.

You can even couple the F.SIGHT single-cell analytical pipelines. It requires lower sample volumes to process than FACS. This allows researchers to isolate cells from limited samples.  

The technology is even able to read low signals. This means that researchers still are able to identify the target population. Even when the fluorescence of the transfected cells is extremely limited. 
 

How they overcame limitations in the workflow

 
The team also used emulsion coupling based on a digital method as an innovative assay in their workflow.

This detects any differences in partitioning of two antibodies labeled with target oligonucleotides in the presence of target proteins compared to their free and unbound distribution in ddPCR emulsion.

Emulsion coupling is homogeneous, molecularly sensitive, and quantitative. It is very specific and readable by sequencing (e.g., next-generation sequencing) in a highly parallel manner. This is due to this assay's two-antibody principle.

This way, the F.SIGHT single-cell fluorescent dispenser became a way of overcoming limitations in the workflow. 

Workflow


  • The team detected Genome-wide changes using a surveyor assay 

  • They monitored Target gene transcript levels= by RT-PCR 

  • They also measured target protein expression by clones by Emulsion coupling 
     
These assays allow the characterization of clones from thousands down to just a few hundred cells. They facilitate low clonal expansion and high characterization throughput while still providing high-quality data about the clones. 

They even facilitate success across cherry-picked clones. You can use the workflow proposed by the team at the University of Freiburg with industrial requirements. It is automatable and you can easily transfer it to other cell types and genetic targets. 
 
You can adapt the workflow for other cell types and genetic targets too. It has great potential for several applications that require monoclonal cells. Plus, this workflow handles different cell types. 

This means that you can say goodbye to any restrictions with single-cell dispensing technologies. 
Emulsions coupling reduces both the time and resources you need to increase the content of characterization workflows.

This allows for better clones with lower failure rates in the final stages of cell line development.


Single cell dispensig


Want to know more? 

Are you interested in knowing more about how the F.SIGHT single cell dispenser can ease your workflow? 

Contact one of our Product Specialists and we will help you find the right solution for you.

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