HomeInVivo Biosystems BlogView from the BenchValidating Expression of Humanizations – What We’ve Done so Far

Validating Expression of Humanizations – What We’ve Done so Far

validating humanizations

In an effort to create better research models for epilepsy, we’ve been working on a grant-funded project to humanize numerous C. elegans genes. We do this by replacing the endogenous coding sequences with human coding sequences (whole gene humanization). Through this experience we have learned a lot about the humanization process and have developed an efficient way to validate our strains. 

When humanizing a C. elegans gene it is key to make sure the human ortholog  expresses and functions similarly to the endogenous gene. Sometimes determining this can be relatively easy. For instance, when the gene you are humanizing has a knockout phenotype (e.g. lethality, movement defects, drug/stress resistance) that is rescued with the humanized protein. However, sometimes the genes you are working with do not have an easily determined phenotype. Or even worse, you have a phenotype but your humanized gene does not rescue the knockout phenotype. In both these cases it is important to validate your humanized gene’s expression. 

There are many methods to test gene expression (northern/western blots, FISH, IHC, microarrays, RNA seq, RT-qPCR) but some of these techniques can be laborious and time consuming. Our lab's go-to method for validating humanized C. elegans genes is a two step RT-qPCR. This technique allows us to quickly assay three critical factors at once. 

  1. First, is the humanized gene being transcribed? This is an important step because the humanization process might disrupt an unexpected transcriptional regulatory element (e.g. alternative start site). 
  2. Second, is the humanized gene being expressed at the same level as the endogenous gene? If the humanized gene is not rescuing the phenotype this could be a key troubleshooting step that will help determine ways to correct the problem.
  3. Third, is the humanized gene splicing correctly? Mis-splicing may lead to the transcript being targeted for nonsense mediated decay. Also, mis-splicing of genes alone has been linked to many human diseases(1) and could possibly explain why a humanized gene does not rescue a phenotype.

By implementing a strain validation process we have been able to find and address unexpected issues that might have confounded experimental data. It has also become a valuable troubleshooting tool for strains that are not behaving as expected.

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