Résumé
Afin de développer des vecteurs d'expression de poisson, entièrement homologues, destinés aux microinjections dans des oeufs fertilisés, les constructions suivantes ont été préparées: promoteurs de la metallothionine, a ou b, de truite arc-en-ciel d'une part, et promoteur de l'actine β de carpe d'autre part, associés à l'ADNc de l'hormone de croissance de daurade royale (ptMTa-gsbGH cDNA, ptMTb-gsbGH cDNA, et pcAβ-gsbGH cDNA).
Les promoteurs de la metallothionine ont été clonés en utilisant la technique de la RCP. La tMTa comprend 430 pb. tandis que la tMTb en comprend 260 (Hong et al. 1992). Ces deux promoteurs ont été insérés dans pGEM-3Z qui contenait l'ADNc de GH de Sparus aurata, pour former, respectivement, ptMTa-gsbGH et ptMTb-gsbGH. Le gène de l'actine cytoplasmique β de carpe été choisi comme source d'isolement de séquences régulatrices fortement constitutives. Une de ces séquences régulatrices a été liguée à l'ADNc de GH de S. aurata dans pUC118, pour réaliser la construction pcAβ-gsbGH cDNA.
L'expression des constructions contenant les promoteurs de la metallothionine a été tentée dans des cultures de cellules de poisson, où elle a été effectivement induite par le zinc. La construction ptMTa-gsbGH cDNA a été microinjectée dans des oeufs fertilisés de carpe. Son intégration dans le génome de carpe a pu être détectée dans l'ADN isolé à partir de nageoires d'animaux agés de 2 mois.
Abstract
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following constructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp β-actin gilthead seabream GH cDNA (pcAβ-gsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter β-actin was isolated from carp.
The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic β-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was ligated to GH cDNA of S. aurata to form the pcAβ-gsbGHcDNA.
Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found to be induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
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Cavari, B., Hong, Y., Funkenstein, B. et al. All-fish gene constructs for growth hormone gene transfer in fish. Fish Physiol Biochem 11, 345–352 (1993). https://doi.org/10.1007/BF00004584
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DOI: https://doi.org/10.1007/BF00004584