Construction and detection of fluorescent, germline transgenic zebrafish

Zebrafish transgenesis was first demonstrated over 10 yr ago; however, the intensity of technological development has significantly increased in the past few years with the popularity of the zebrafish model. By transgenesis we are referring to germline transgenesis-the introduction of new genetic material into the germline, such that it is passed down to progeny and inherited in a Mendelian manner. This chapter describes the procedures used to produce transgenic zebrafish expressing fluorescent reporter genes via DNA microinjection. These procedures were modified from those developed by members of this laboratory during the establishment of the transgenic mouse facility at Duke Comprehensive Cancer Center. This chapter aims to aid others in efficiently producing transgenic zebrafish. The development of transgenic technology in zebrafish appears to have mirrored the early development of that in mice. Observations and manipulations from several different laboratories were necessary to determine conditions that would routinely yield detectable transgene expression in mice. Two fundamental modifications that facilitated the generation of transgene-expressing mice are (1) the elimination of plasmid sequences from the introduced transgene, because these sequences could interfere with or provide faulty expression patterns of the transgene; and (2) the addition of at least one intron to the transgenic construct. These same modifications have proven useful in the generation of transgene-expressing zebrafish. As with zebrafish, some of the first transgenes introduced into mouse embryos were viral in origin. Both SV40 and Moloney murine leukemia virus (MMLV) were introduced into the mouse germline. However, it became clear that these viral sequences were not transcriptionally adequate in either the early mouse embryos or in the early embryonal carcinoma cells. As mouse genes and promoters were identified, the field of expression transgenesis developed rapidly and resulted in transgenic mouse facilities being commonplace on most university campuses. Presently, the introduction of ectopic expression via the germline is being eclipsed by technology in which embryonic stem (ES) cells that carry specific mutations or deletions within selected genes are reintroduced to generate knockout mice. A similar technology for zebrafish is currently under analysis by Collodi and colleagues.