PUBLICATION
A reverse genetic approach to test functional redundancy during embryogenesis
- Authors
- Rikin, A., Rosenfeld, G.E., McCartin, K., and Evans, T.
- ID
- ZDB-PUB-100826-22
- Date
- 2010
- Source
- Journal of visualized experiments : JoVE (42): (Journal)
- Registered Authors
- Evans, Todd
- Keywords
- none
- MeSH Terms
-
- Animals
- Embryo, Nonmammalian
- Embryonic Development/genetics*
- Female
- GATA Transcription Factors/genetics
- GATA5 Transcription Factor/genetics
- Gene Knockdown Techniques/methods*
- Male
- Oligonucleotides, Antisense/genetics
- Zebrafish/embryology*
- Zebrafish/genetics*
- Zebrafish Proteins/genetics
- PubMed
- 20736915 Full text @ J. Vis. Exp.
Citation
Rikin, A., Rosenfeld, G.E., McCartin, K., and Evans, T. (2010) A reverse genetic approach to test functional redundancy during embryogenesis. Journal of visualized experiments : JoVE. (42).
Abstract
Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping