Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Abstract

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox with diverse applications in vertebrate model systems

“…While Tol2-mediated transposition has been a mainstay for more than 25 years for generating transient and stable transgenic lines, the I-SceI meganuclease system is equally effective for generating transient and stable transgenic animals by circumventing the limited persistence of episomal plasmid DNA following multiple rounds of cell division and overcomes low germline transmission rates due to unequal integration of episomal DNA fragments into the host genome [144][145][146][147][148][149] . I-SceI, an intron-mediated homing endonuclease isolated from yeast 150,151 , has an 18-bp recognition sequence expected to occur only once in 7x10 10 bases of random sequence and is unlikely to cut genomic DNA.…”

Transposon-mediated transgenesis in the frog: New tools for biomedical and developmental studies

“…A number of techniques to create transgenic frogs have been developed in the past two decades to incorporate reporter genes into the frog genome. Larry Etkin and coworkers were the first to successfully generate transgenics by injecting linear DNA, in this case a chloramphenicol reporter gene, into X. laevis embryos (12). The injected embryos were mosaic and founder animals carrying the transgene were generated in limited numbers.…”