Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos

Abstract

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.

5′-Modifications improve potency and efficacy of DNA donors for precision genome editing

“…In addition, due to their size, long donor molecules may not transit the nuclear envelope as efficiently, reducing the effective concentration at the site of repair, or requiring cell division to gain access to the target locus. Moreover, end-joining ligation reactions assemble linear dsDNA molecules into concatemers in eukaryotic cells ( Perucho et al, 1980 ; Folger et al, 1982 ; Mello et al, 1991 ; Stuart et al, 1988 ; Lacy et al, 1983 ), further limiting the number of individual donor molecules and their ability to diffuse to their DSB target sites.…”

The Role of Macrophages During Zebrafish Injury and Tissue Regeneration Under Infectious and Non-Infectious Conditions

“…Importantly, the zebrafish genome shares 70% homology with the human genome, offering the possibility to study a wide panel of human diseases such as muscular dystrophies, cardiovascular diseases or infectious diseases. Zebrafish represent a widely used model to investigate regenerative mechanisms of a large diversity of tissues and organs (liver, pancreas, jaw, lateral line's hair cells, retina, heart, central nervous system and caudal fin) throughout their various developmental stages (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

Progress in Gene-Editing Technology of Zebrafish

“…In the 1980s, with the successful application of transgenic technology in fish, a milestone in fish gene-editing technology was achieved [3]. Originally, the target gene, including plasmid DNA and bacterial artificial chromosomes, was introduced into the organism to meet human demands [4,5]. Traditionally, linearized exogenous DNA was injected into fertilized eggs alone to realize the transgene.…”

“…Traditionally, linearized exogenous DNA was injected into fertilized eggs alone to realize the transgene. However, the integration efficiency of foreign genes into the genome and the probability of transmitting the DNA to offspring is very low [5]. Moreover, the technique is non-directional, and the presence of multi-exogenous plasmid copies may lead to abnormal development of fertilized eggs, a large number of malformed offspring, and difficulty in integration site detection.…”