Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors.
In the chick embryo, two methods are now used for studying the developmental role of genes by loss-of-function approaches: vector-based shRNA and morpholino oligonucleotides. Both have the advantage that loss-of-function can be conducted in a spatially and temporally controlled way by focal electroporation. Here, we compare these two methods. We find that the shRNA expressing vectors pRFPRNAi, even when targeting a non-expressed protein like GFP, cause morphological phenotypes, mis-regulation of non-targeted genes and activation of the p53 pathway. These effects are highly reproducible, appear to be independent of the targeting sequence and are particularly severe at primitive streak and early somite stages. By contrast, morpholinos do not cause these effects. We propose that pRFPRNAi should only be used with considerable caution and that morpholinos are a preferable approach for gene knock-down during early chick development.
Mende
Michael
M
Department of Craniofacial Development, King's College London, Guy's Campus, Guy's Tower Floor 27, London SE1 9RT, UK.
Christophorou
Nicolas A D
NA
Streit
Andrea
A
D000818:Animals; D002642:Chick Embryo; D017931:DNA Primers / Q000737:chemistry; D018274:Electroporation; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques / Q000295:instrumentation / Q000379:methods*; D020868:Gene Silencing; D016158:Genes, p53; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry*; D008954:Models, Biological; D009841:Oligonucleotides / Q000235:genetics; D034622:RNA Interference; D000097573:Red Fluorescent Protein
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009841:Oligonucleotides; D000097573:Red Fluorescent Protein