Transgene manipulation in rainbow trout using Cre recombinase

Sachi Kume, Naoto Katayama, Kensuke Ichida, Shoko Hattori-Ihara, Kazue Nagasawa, Goro Yoshizaki

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.

Original languageEnglish
Pages (from-to)767-773
Number of pages7
JournalFisheries Science
Volume80
Issue number4
DOIs
Publication statusPublished - 2014 Jan 1
Externally publishedYes

Keywords

  • Cre recombinase
  • GFP
  • Rainbow trout
  • Transgenic fish
  • loxP

ASJC Scopus subject areas

  • Aquatic Science

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