Development of cell cultures with competency for contributing to the zebrafish germ line.

The zebrafish is an established model for the genetic analysis of vertebrate development. Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis. Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish. Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines. Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures. Several strategies are being used to optimize the production and identification of germ-line chimeras. The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.

Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture.

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term primary embryo cell cultures. Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo, including muscle, liver, gut, and fin. To facilitate the identification of germ-line chimeras, ES cells expressing the green fluorescent protein (GFP) were introduced into host embryos, and germ-line contribution was detected by the presence of GFP+ cells in the region of the gonad. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.

Spontaneous atherosclerosis in the proximal aorta of LPA transgenic mice on a normal diet.

Serum levels of Lp(a) lipoprotein are under genetic control and a high level is a risk factor for atherosclerotic disease. We have examined the aorta of LPA transgenic mice and their non-transgenic litter mates who had all been given a regular, not lipid fortified diet. When sacrificed, the animals had an average age of 66 weeks. Lipid lesions were observed in the aorta of 13 out of 18 LPA transgenic mice and in five out of 21 non-transgenic animals. The difference is statistically significant. We conclude that LPA transgenic mice develop lipid lesions in aorta more frequently than non-transgenic animals, even on a diet with a low fat content. LPA transgenic mice on a normal diet could be a useful animal model for the study of spontaneous human atherosclerosis, its treatment and prevention.