Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis.
Nat Protoc
; 8(5): 924-34, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23579781
ABSTRACT
We describe a protocol for microinjection of embryos for an emerging model system, the cnidarian sea anemone, Nematostella vectensis. In addition, we provide protocols for carrying out overexpression and knockdown of gene function through microinjection of in vitro-translated mRNAs or gene-specific oligonucleotide morpholinos (MOs), respectively. Our approach is simple, and it takes advantage of the natural adherence properties of the early embryo to position them in a single layer on a polystyrene dish. Embryos are visualized on a dissecting microscope equipped with epifluorescence and injected with microinjection needles using a picospritzer forced-air injection system. A micromanipulator is used to guide the needle to impale individual embryos. Injection takes â¼1.5 h, and an experienced researcher can inject â¼2,000 embryos in a single session. With the availability of the published Nematostella genome, the entire protocol, including cloning and transcription of mRNAs, can be carried out in â¼1 week.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Anêmonas-do-Mar
/
RNA Mensageiro
/
Morfolinos
/
Genética Reversa
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article