The recent advancements of gene-editing technologies, such as CRISPR, has enabled scientists to create more precise zebrafish models of human disease to use in their research. While there have been many advancements, zebrafish knock-in (KI) techniques still aren’t fully optimized or understood. In this article, we aim to give a quick summary of the different types of knock-in tagging techniques.
Knock-in Tagging Techniques:
Through knock-in tagging technology, you can tag your favorite gene at the native locus in your zebrafish line via CRISPR/Cas9 gene editing.
There are two main knock-in tagging techniques, knocking in a fluorescent tag at the native locus and knocking in a protein tag. Fluorescent tagging is typically used to aid in visualization of spatiotemporal dynamics of a given gene's protein product in live (and sometimes fixed) animals. Epitope tagging is most often utilized to facilitate biochemical studies, in particular for studies of protein-protein interactions which require the presence of a readily recognizable epitope or sequence feature for chemical modification or isolation.
Both techniques have slightly different benefits, so choosing the correct one for your research is important. If you are ever in doubt, one of our zebrafish experts is happy to discuss which options is right for you.
- Knocking in a fluorescent tag at the native locus. Observe when and where your gene of interest is expressed using fluorescence. A genetically encoded fluorescent marker (GFP, mCherry, etc.) can be inserted in-frame for tagging of either the N- or C-terminus of your protein.
- Knocking in a protein tag. Genetically encoded tags (FLAG tag, HA-tag, poly(His) tag, etc.) can be inserted in-frame for tagging of either the N- or C-terminus of your protein. The protein can then be observed by Western blots, immunofluorescence, and immunoprecipitation using commercially available common antibodies.
Fluorescent Tagging vs. Epitope Tagging in Zebrafish
Not sure which option is right for you? We created a simple breakdown of the applications, pros, and cons of each technique so you can help guide yourself to the right knock-in technique for you.
|Fluorescent Tagging||Epitope Tagging|
|Example of tags|
in fixed tissue samples
|Pros||Can aid in visualization of spatiotemporal dynamics of a given gene's protein product in live (and sometimes fixed) animals.||Can facilitate visualization or isolation of protein expression with
reduced chance of disrupting endogenous expression levels or
protein trafficking dynamics due to relatively small size.
Can also facilitate visualization or biochemical modification of proteins in fixed tissue with comparatively robust and cheap commercially available antibodies.
|Cons||Can interfere with gene function due to the relatively large size of fluorophores used.||Not sufficiently validated. Lack of publications.|
User Showcae: Dr. Melissa A. Wright
Dr. Wright studies the SELENON gene (also called SEPN1) which encodes a protein called selenoprotein N, and she uses zebrafish as the model organism to better understand its function.
(Left): SelN-BFPSTOP with BFP followed by a STOP codon inserted into the N-terminus of Selenon (SelN). (Center and Right): Ryr1a-mCherry line where the endogenous Ryr1a was tagged with mCherry at the N-terminus. Image courtesy of Melissa A. Wright, MD/PhD, Assistant Professor of Pediatric Neurology at University of Colorado.
Selenoprotein N is highly active in many tissues before birth and may be involved in the formation of muscle tissue (myogenesis). The protein may also be important for normal muscle function after birth, although it is active at much lower levels in adult tissues.
The exact function of selenoprotein N is unknown, it is likely involved in protecting cells against oxidative stress. Oxidative stress occurs when unstable molecules called free radicals accumulate to levels that damage or kill cells.
Though CRISPR/Cas9 gene editing in zebrafish has gained popularity, it is not without its technological barriers. Reagent sourcing, sg design and validation of cutting efficiency, and donor homology design are challenging, and can often be a lengthy process without a guarantee of success even for highly skilled researchers.
Many investigators choose to use customized injection mix in order to quickly start their project. It is also a good solution for those with a more limited budget who want to utilize CRISPR technology to create novel zebrafish lines. Dr. Wright used our injection mix to jump start her project by leveraging our design expertise in order to save time and get results more quickly.