Improving the production of transgenic fish germlines: in vivo evaluation of mosaicism in zebrafish (Danio rerio) using a green fluorescent protein (GFP) and growth hormone cDNA transgene co-injection strategy

Abstract

In fish, microinjection is the method most frequently used for gene transfer. However, due to delayed transgene integration this technique almost invariably produces mosaic individuals and if the gene is not integrated into germ cells its transmission to descendants is difficult or impossible. We evaluated the degree of in vivo mosaicism using a strategy where a reporter transgene is co-injected with a transgene of interest so that potential germline founders can be easily identified. Transgenic zebrafish (Danio rerio) were produced using two transgenes, both comprised of the carp β-actin promoter driving the expression of either the green fluorescent protein (GFP) reporter gene or the growth hormone cDNA from the marine silverside fish Odonthestes argentinensis. The methodology applied allowed a rapid identification of G 0 transgenic fish and also detected which fish were transmitting transgenes to the next generation. This strategy also allowed inferences to be made about genomic transgene integration events in the six lineages produced and allowed the identification of one lineage transmitting both transgenes linked on the same chromosome. These results represent a significant advance in the reduction of the effort invested in producing a stable genetically modified fish lineage.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

“…Therefore, the presence of a higher level of exogenous GH in transgenic animals could lead to metabolic alterations that can influence not only the animal growth, but also several other traits. During the last decade, the interest on GH overexpression effects has increased significantly, and several transgenic animal models overexpressing GH have been developed, including mammals (mouse and rat) and some teleost fishes (Oncorhynchus mykiss, Danio rerio) (Ikeda et al 1998, Devlin et al 2001, Lall et al 2004, Figueiredo et al 2007. However, there is few data on the GH overexpression effects in brain, a feature that has only been clearly analyzed using mouse GH transgenic models (Lall et al 2004, Bohlooly-Y et al 2005.…”

“…The GH transgenic zebrafish belong to the F0104 line that has a carp (Cyprinus carpio) β-actin promoter driving the expression of two exogenous genes -the growth hormone gene from the marine silverside fish (msGH) Odonthestes argentinensis and the jellyfish (Aequorea victoria) GFP (Green Fluorescent Protein) as a reporter gene to monitor for transformed zygotes (Figueiredo et al 2007). Transgenic and NT genotypes were determined through UV fluorescence analysis with an epifluorescence microscope (excitation=485 nm; emission=520 nm) through the presence or absence of a fluorescence level related to the GFP expression.…”

Effects of Double Transgenesis of Somatotrophic Axis (GH/GHR) on Skeletal Muscle Growth of Zebrafish (Danio rerio)

“…All analyzed genotypes were obtained by breeding males from the F0104 line 11 with females from the MYO-GHR line. 12 Besides GH, the F0104 line also expresses the green fluorescent protein (GFP) under transcriptional control of the b-actin promoter of carp, Cyprinus carpio.…”

TRANSMISI, EKSPRESI, DAN DISTRIBUSI GEN HORMON PERTUMBUHAN IKAN PATIN SIAM PADA IKAN LELE AFRIKA (Clarias gariepinus) TRANSGENIK F-2

“…Hasil yang berbeda dilaporkan oleh Rahman & Maclean (1999), menyatakan bahwa ikan nila F-2 yang membawa gen hormon pertumbuhan yang dihasilkan dari perkawinan transgenik F-1 dengan non-transgenik menghasilkan rata-rata transmisi transgen sebesar 50%. Figueiredo et al (2007), melaporkan bahwa ikan zebra F-2 yang membawa gen hormon pertumbuhan yang merupakan hasil persilangan transgenik F-1 dengan non-transgenik juga menghasilkan transmisi transgen pada F-2 sebesar 50%. Stuart et al (1990), juga melaporkan bahwa transmisi transgen yang membawa plasmid pUSVCAT pada ikan zebra dari F-1 ke F-2 berkisar 48%-56%.…”