Transgenic fish with tissue-specific expression

Abstract

Disclosed are transgenic fish, and a method of making transgenic fish, which express transgenes in stable and predictable tissue- or developmentally-specific patterns. The transgenic fish contain transgene constructs with homologous expression sequences. Also disclosed are methods of using such transgenic fish. Such expression of transgenes allow the study of developmental processes, the relationship of cell lineages, the assessment of the effect of specific genes and compounds on the development or maintenance of specific tissues or cell lineages, and the maintenance of lines of fish bearing mutant genes.

I claim:  
     
         1 . A transgenic fish the cells of which contain an exogenous construct, wherein the construct comprises homologous expression sequences operably linked to a sequence encoding an expression product, wherein the expression product is expressed only in specific cell lineages.  
     
     
         2 . The transgenic fish of  claim 1  wherein the expression sequences and the sequence encoding the expression product are not operably linked in nature.  
     
     
         3 . The transgenic fish of  claim 1  wherein the expression product is heterologous.  
     
     
         4 . The transgenic fish of  claim 3  wherein the expression product is a reporter protein.  
     
     
         5 . The transgenic fish of  claim 4  wherein the reporter protein is selected from the group consisting of β-galactosidase, chloramphenicol acetyltransferase, and green fluorescent protein.  
     
     
         6 . The transgenic fish of  claim 5  wherein the reporter protein is green fluorescent protein.  
     
     
         7 . The transgenic fish of  claim 1  wherein the fish is selected from the group consisting of zebrafish, medaka, trout, salmon, carp, tilapia, goldfish, loach, and catfish.  
     
     
         8 . The transgenic fish of  claim 7  wherein the fish is zebrafish.  
     
     
         9 . The transgenic fish of  claim 1  wherein the expression product is expressed only in cells selected from the group consisting of blood cells, nerve cells, and skin cells.  
     
     
         10 . The transgenic fish of  claim 9  wherein the expression product is expressed only in blood cells.  
     
     
         11 . The transgenic fish of  claim 10  wherein the expression product is expressed only in erythroid progenitor cells.  
     
     
         12 . The transgenic fish of  claim 9  wherein the expression product is expressed only in neurons.  
     
     
         13 . The transgenic fish of  claim 1  wherein the expression sequences are selected from the group consisting of GATA-1 expression sequences and GATA-2 expression sequences.  
     
     
         14 . The transgenic fish of  claim 13  wherein the expression sequences comprise GATA-1 expression sequences.  
     
     
         15 . The transgenic fish of  claim 13  wherein the expression sequences comprise GATA-2 expression sequences.  
     
     
         16 . The transgenic fish of  claim 15  wherein the expression sequences comprise the GATA-2 promoter operably linked to the neuron-specific enhancer of GATA-2.  
     
     
         17 . The transgenic fish of  claim 15  wherein the expression sequences comprise the GATA-2 promoter operably linked to the blood-specific enhancer of GATA-2.  
     
     
         18 . The transgenic fish of  claim 15  wherein the expression sequences comprise the GATA-2 promoter operably linked to the skin-specific enhancer of GATA-2.  
     
     
         19 . The transgenic fish of  claim 1  wherein the transgenic fish developed from, or is the progeny of a transgenic fish developed from, an embryonic cell into which the construct was introduced.  
     
     
         20 . The transgenic fish of  claim 1  wherein the expression product is expressed only in predetermined cell lineages.  
     
     
         21 . The transgenic fish of  claim 1  wherein the exogenous construct is genetically linked to an identified mutant gene.  
     
     
         22 . The transgenic fish of  claim 1  wherein the expression sequences comprise a homologous promoter operably linked to a homologous enhancer.  
     
     
         23 . The transgenic fish of  claim 22  wherein the expression sequences further comprise homologous 5′ untranslated sequences operably linked to the promoter and the sequence encoding the expression product.  
     
     
         24 . The transgenic fish of  claim 1  wherein the construct further comprises (a) intron sequences operably linked to the sequence encoding the expression product, (b) a polyadenylation signal operably linked to the sequence encoding the expression product, or both.  
     
     
         25 . Cells isolated from the transgenic fish of  claim 1  wherein the cells express the expression product.  
     
     
         26 . A method of making transgenic fish, the method comprising 
 (a) introducing an exogenous construct into an embryonic cell of a first fish, wherein the construct comprises homologous expression sequences operably linked to a sequence encoding an expression product, and    (b) allowing the egg cell or embryonic cells to develop into a second fish, wherein the expression product is expressed only in specific cell lineages of the second fish.    
     
     
         27 . The method of  claim 26  wherein the expression product is expressed only in predetermined cell lineages.  
     
     
         28 . The method of  claim 26  wherein the method further comprises producing progeny of the second fish.  
     
     
         29 . The method of  claim 26  wherein the expression sequences and the sequence encoding the expression product are not operably linked in nature.  
     
     
         30 . The method of  claim 26 , wherein the expression sequences are expression sequences of a fish gene, wherein the method further comprises 
 (c) exposing the second fish or progeny of the second fish to a test compound,    (d) detecting the expression product in the fish exposed to the test compound, and    (e) comparing the pattern of expression of the expression product in the fish exposed to the test compound with the pattern of expression of the expression product in the second fish or progeny of the second fish not exposed to the test compound,    wherein if the pattern of expression of the expression product in the fish exposed to the test compound differs from the pattern of expression in the fish not exposed to the test compound, then the test compound affects expression of the fish gene.    
     
     
         31 . The method of  claim 26 , wherein the expression sequences are expression sequences of a fish gene, wherein the method further comprises 
 (c) detecting the expression product in the second fish or progeny of the second fish,    wherein the pattern of expression of the expression product in the second, fish or progeny of the second fish identifies the pattern of expression of the fish gene.    
     
     
         32 . The method of  claim 26 , wherein the expression sequences are expression sequences of a fish gene, wherein the method further comprises 
 (c) crossing the second fish or progeny of the second fish to a third fish having an identified mutant gene to produce a fourth fish having both the exogenous construct and the identified mutation,    (d) detecting the expression product in the fourth fish or progeny of the fourth fish, and    (e) comparing the pattern of expression of the expression product in the fourth fish or the progeny of the fourth fish with the pattern of expression of the expression product in the second fish,    wherein if the pattern of expression of the expression product in the fourth fish or progeny of the fourth fish differs from the pattern of expression in the second fish, then the mutant gene affects expression of the fish gene.    
     
     
         33 . The method of  claim 26 , wherein the method further comprises 
 (c) crossing the second fish or progeny of the second fish to a third fish having an identified mutant gene, wherein the exogenous construct and the mutant gene map to the same region of the genome, to produce a fourth fish having both the exogenous construct and the mutant gene, and    (d) crossing the fourth fish to a fifth fish, wherein the fifth fish has neither the exogenous construct nor the mutant gene, to produce a sixth fish, wherein the sixth fish has both the exogenous construct and the mutant gene,    wherein the mutant gene is marked by the exogenous construct in the sixth fish.    
     
     
         34 . The method of  claim 33 , wherein the method further comprises 
 (e) crossing the sixth fish, or a progeny of the sixth fish, with a seventh fish, and    (f) identifying progeny fish expressing the expression product, wherein fish expressing the expression product have the mutant gene.    
     
     
         35 . The method of  claim 26 , wherein the construct comprises a homologous promoter operably linked to a sequence encoding an expression product, wherein the promoter is not operably linked to a enhancer, wherein the method further comprises 
 (c) detecting the expression product in the second fish or progeny of the second fish,    wherein if the expression product is detected, then the exogenous construct is operably linked to a enhancer.    
     
     
         36 . The method of  claim 35  further comprising 
 (d) isolating the enhancer from the second fish or progeny of the second fish.  
 
     
     
         37 . The method of  claim 35  further comprising 
 (d) determining the pattern of expression of the expression product in the second fish or progeny of the second fish,  
 wherein the pattern of expression of the expression product in the second fish or progeny of the second fish identifies the pattern of expression of the enhancer.  
 
     
     
         38 . A method of identifying regulatory elements in sequences upstream of a gene of interest, the method comprising 
 (a) introducing members of a set of exogenous constructs into separate embryonic cells, wherein each member of the set of constructs comprises a sequence encoding an expression product operably linked to upstream sequences of a homologous gene of interest, wherein the different members of the set have different regions of the upstream sequences deleted,    (b) allowing the embryonic cells to develop into fish,    (c) detecting the expression product in the fish or progeny of the fish,    (d) determining which regions of the upstream sequences are needed for expression of the expression product.    
     
     
         39 . The method of  claim 38  wherein determining which regions of the upstream sequences are needed for expression is accomplished by comparing the expression of the expression product in fish into which different members of the set of exogenous constructs has been introduced, 
 wherein if the expression product is detected in cells of interest in a fish, then the exogenous construct introduced into that fish includes a regulatory element for expression in the cells of interest,  
 wherein if the expression product is not detected in cells of interest in a fish, then the exogenous construct introduced into that fish does not include a regulatory element for expression in the cells of interest.  
 
     
     
         40 . A nucleic acid construct comprising expression sequences derived from fish operably linked to a sequence encoding an expression product, wherein the expression sequences comprise a promoter operably linked to a enhancer, wherein the expression product is expressed only in specific cell lineages.