A Novel Zebrafish Model for Assessing In Vivo Delivery of Morpholino Oligomers

Morpholino oligomers have great therapeutic potential for treatment of a broad range of human diseases, including viral, bacterial, age-related, and genetic diseases, but they suffer from poor systemic delivery into cells. Although various approaches have been undertaken to address the delivery problem, it remains as the major barrier of morpholinos to be used as effective therapeutics. This slow development is in part due to the cost of materials and the animal models used for screening the efficacy and safety of those delivery approaches. The need to have an inexpensive vertebrate model for assessing in vivo delivery of morpholinos is evident. Therefore, we have produced a novel transgenic zebrafish model containing a dual reporter cassette for determination of in vivo delivery, bio-distribution, and safety of a morpholino. The levels of morpholino delivered to the cells in various tissues can be determined by changes in reporter gene expressions caused by morpholino-induced exon skipping. This chapter provides a description of the reagents, equipment, and procedure for successful retro-orbital injection of a peptide-conjugated morpholino into the blood stream of the adult zebrafish to cause targeted exon skipping in the heart of the zebrafish.