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transient-overexpression-mRNA-injection Transient Overexpression

Several pathological conditions are caused by aberrant protein expression. To characterize the effect of abnormal protein expression, it is possible to employ transient overexpression models.

Moreover, a similar strategy can be employed to validate the pathogenetic role (or the biological function) of a protein. Indeed, overexpression experiments can be employed to rescue the phenotypes observed in a loss-of-function model. The injection of an mRNA encoding for the coding sequence (CDS) of interest can be employed to ubiquitously overexpress a protein during early zebrafish development (up to 3 days). Alternatively, to investigate the effect of protein overexpression at later stages or in restricted cell types, it is possible to inject a plasmid in which the CDS of interest is preceded by a ubiquitous or a tissue-specific promoter. 

The expression of the exogenous CDS can be associated with one fluorescent reporter to facilitate the phenotypic analysis of the models generated.

Applications

  • Temporary overexpression (at early developmental stages or in tissues of interest)
  • Cost-effective screening of candidate genes whose increased expression is supposed to have a pathological role
  • Rescue of phenotypes associated to gene loss-of-function 
  • Evaluation of mRNA toxicity at different concentrations

Advantages

Fast target validation prior to the generation of stable transgenic lines

High throughput screening of multiple candidate genes

Non-invasive, in vivo readouts to detect overexpression-induced phenotypes at the level of cells, tissues or organs

Method description

To generate a model based on mRNA’s expression, zebrafish one-cell-stage embryos are injected with a solution containing the mRNA synthesized in vitro and encoding for the CDS of interest. 

For plasmid overexpression, a donor plasmid containing the exogenous CDS of interest is injected into one-cell-stage embryos. During early stages of embryonic development, the plasmid can be imported in the nucleus and remain episomic. The exogenous CDS can be expressed by the promoter sequence included in the plasmid giving rise to F0 larvae transiently expressing the sequence of interest.

Figure 1.  Transient overexpression in embryos injected with an mRNA encoding for the gene of interest. Effect of mRNA expression can be readily analyzed during early development.

Readouts

Results of the phenotypic analysis according to customers' interests and needs:

  • Teratogenic phenotypes
  • Organ-specific morphological or functional defects (heart-specific, liver-specific etc.)
  • Behavioral phenotypes

References

  1. Liu Y, Lin Z, Liu M, Wang H, Sun H. Overexpression of DYRK1A, a Down Syndrome Candidate gene, Impairs Primordial Germ Cells Maintenance and Migration in zebrafish. Sci Rep. 2017 Nov 10;7(1):15313.
  2. Finckbeiner S, Ko PJ, Carrington B, Sood R, Gross K, Dolnick B, Sufrin J, Liu P. Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene. Cell Biosci. 2011 Sep 26;1:32.
  3. Hogan BM, Verkade H, Lieschke GJ, Heath JK. Manipulation of gene expression during zebrafish embryonic development using transient approaches. Methods Mol Biol. 2008;469:273-300.