Transgene manipulation in rainbow trout using Cre recombinase
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 th...
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Published in | Fisheries science Vol. 80; no. 4; pp. 767 - 773 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Tokyo
Springer Japan
01.07.2014
Springer Nature B.V |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0919-9268 1444-2906 |
DOI: | 10.1007/s12562-014-0742-x |