Novel vector systems optimized for injecting in vitro-synthesized mRNA into zebrafish embryos

Microinjection of nucleic acids or proteins is a useful way of studying embryonic development. In particular, injection of in vitro-transcribed capped RNA is commonly employed to achieve ectopic or increased expression of genes. Two vector systems, pCS2+ and pT7Ts, have been used for this purpose in zebrafish. However, they were initially optimized for Xenopus embryos not for zebrafish. Here we describe a vector, pcGlobin2, optimized for zebrafish, and its derivative, pcGlobin2-GST. This new vector system offers several advantages. First, pcGlobin 2 contains three critical elements 15' and 3' zebrafish beta-globin UTRs, and a poly(A) tail] for generating stable mRNAs and greatly improving translation efficiency. Second, subcloning and preparation of template DNA is easier because of the larger number of restriction sites. Third, protein-binding assays can be performed directly on the injected embryos with pcGlobin2-GST. Lastly, this vector system can be transfected into animal cells without additional subcloning.