Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Cartilaginous fishes have various unique physiological features such as cartilaginous skeletons and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are considered a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, experimental approaches are limited in cartilaginous fishes. Particularly, genetic engineering, which can analyze gene functions as well as cellular behavior, has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Trials of gene transfer have also been limited both in in vitro cultured cells and in vivo. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene introduction. First, we established a primary cell culture containing fibroblast-like and epithelial-like cells from cloudy catshark embryos. Using these primary cultured cells, we attempted gene transfection by lipofection, polyethylenimine (PEI), adenovirus, baculovirus and electroporation. Among the methods tested, lipofection, electroporation and baculovirus infection enabled the successful introduction of exogenous genes into primary cultured cells, allowing us to study physiological mechanisms at a single-cell level in culture conditions close to those in a living cartilaginous fish. We also attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into various tissues including muscle cells. Furthermore, we succeeded in GFP introduction into adult testis by electroporation. The in vitro and in vivo gene introduction methods that worked in this study may identify paths for future genetic manipulation including knockout experiments and cellular linage analysis in cartilaginous fishes.