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.

1 . A transgenic fish that expresses an expression product, comprising a homologous cell lineage-specific expression sequence operably linked to an exogenous nucleic acid sequence encoding the expression product, wherein the cell lineage expression sequence and the exogenous sequence encoding the expression product are integrated into the genome of the fish, wherein the expression product encoded by the transgenic sequence exhibits cell lineage-specific expression, and wherein the fish is selected from the group consisting of salmon, trout, tuna, halibut, catfish, zebrafish, medaka, carp, tilapia, goldfish and loach. 
     
     
         2 . The transgenic fish of  claim 1  wherein the expression sequence 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 . (canceled) 
     
     
         8 . The transgenic fish of  claim 1  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 - 12 . (canceled) 
     
     
         13 . The transgenic fish of  claim 1  wherein the expression sequence is selected from the group consisting of GATA-1 expression sequence and a GATA-2 expression sequence. 
     
     
         14 - 18 . (canceled) 
     
     
         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 sequence encoding the expression product was introduced. 
     
     
         20 . The trangenic fish of  claim 1  wherein the expression product is expressed only in predetermined cell lineages. 
     
     
         21 . The transgenic fish of  claim 1  wherein the sequence encoding the expression product is genetically linked to an identified mutant gene. 
     
     
         22 . The transgenic fish of  claim 1  wherein the expression sequence comprises a homologous promoter operably linked to a homologous enhancer. 
     
     
         23 . (canceled) 
     
     
         24 . The transgenic fish of  claim 1  further comprising (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 a transgenic fish, the method comprising (a) introducing an exogenous construct into an egg cell or embryonic cell of a fish, wherein the construct comprises a homologous cell lineage-specific expression sequence operably linked to a sequence encoding an expression product, and (b) allowing the egg cell or embryonic cell to develop into a fish, (c) crossing the fish of (b) with a second fish to produce a third fish having the exogenous construct, wherein the expression product exhibits cell lineage-specific expression in the third fish and wherein the sequence encoding the expression product is integrated into the genome of the third fish, and wherein the fish is selected from the group consisting of salmon, trout, tuna, halibut, catfish, zebrafish, medaka, carp, tilapia, goldfish and loach. 
     
     
         27 - 29 . (canceled) 
     
     
         30 . A method of identifying a test compound that affects expression or the pattern of expression of a fish gene comprising (a) exposing the fish or transgenic progeny of the fish made by the method of  claim 26  to a test compound, (b) detecting the expression product in the fish exposed to the test compound, and (c) comparing the expression or pattern of expression of the expression product in the fish exposed to the test compound with the expression or pattern of expression of the expression product in the fish or progeny of the fish not exposed to the test compound, wherein if the expression or pattern of expression of the expression product in the fish exposed to the test compound differs from expression or the pattern of expression in the fish not exposed to the test compound, then the test compound affects expression or the pattern of expression of the fish gene. 
     
     
         31 . A method of identifying the pattern of expression of a fish gene comprising detecting the expression product in the fish or transgenic progeny of the fish made by the method of  claim 26 , wherein the pattern of expression of the expression product in the fish or progeny of the fish identifies the pattern of expression of the fish gene. 
     
     
         32 . A method of identifying a mutant gene that affects expression or the pattern of expression of a fish gene comprising (a) crossing the fish or transgenic progeny of the fish made by the method of  claim 26  to a second fish having an identified mutant gene to produce a third fish having both the exogenous construct and the identified mutation, (b) detecting the expression product in the third fish or progeny of the third fish, and (c) comparing the expression or pattern of expression of the expression product in the third fish or the progeny of the third fish with the expression or pattern of expression of the expression product in the second fish, wherein if the expression or pattern of expression of the expression product in the third fish or progeny of the third fish differs from the expression or pattern of expression in the second fish, then the mutant gene affects expression or the pattern of expression of the fish gene. 
     
     
         33 . A method of marking a mutant gene comprising (a) crossing the fish or transgenic progeny of the fish made by the method of  claim 26  to a second 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 third fish having both the exogenous construct and the mutant gene, and (b) crossing the third fish to a fourth fish, wherein the fourth fish has neither the exogenous construct nor the mutant gene, to produce a fifth fish, wherein the fifth fish has both the exogenous construct and the mutant gene, wherein the mutant gene is marked by the exogenous construct in the fifth fish. 
     
     
         34 - 40 . (canceled) 
     
     
         41 . The transgenic fish of  claim 1  wherein expression of the expression product is stable and transmitted through the germline. 
     
     
         42 . The transgenic fish of  claim 1 , wherein the homologous expression sequence and the sequence encoding the expression product are contained in an exogenous construct. 
     
     
         43 . A method of identifying genes, the method comprising:
 (a) isolating from the fish of  claim 1  or progeny thereof a cell expressing the cell lineage-specific expression product, and   (b) identifying genes that are expressed in the cell   
     
     
         44 . The method of  claim 43 , wherein the gene is identified by extracting RNA and performing differential display. 
     
     
         45 . The method of  claim 43 , wherein the gene is identified by extracting RNA and performing subtractive hybridization. 
     
     
         46 . A method for identifying genes involved in cell lineage specific expression comprising:
 (a) Introducing a mutation into the fish of  claim 1  and   (b) Detecting a change in the expression or pattern of expression of the expression product in the fish containing the mutation, whereby a change in the expression or pattern of expression product identifies a gene involved in cell lineage-specific expression.   
     
     
         47 . A method of identifying compounds that affect expression of genes comprising:
 (a) Contacting a first fish of  claim 1  with a compound, wherein the expression product is a directly detectable reporter protein;   (b) Comparing the expression or pattern of expression of the expression product in the fish contacted with the compound with a second fish of  claim 1  that was not contacted with the compound;   (c) Determining the effect of the compound on the expression or pattern of expression of expression product wherein an effect on the expression or pattern of expression of the expression product indicates that the compound affects expression of genes.   
     
     
         48 . The transgenic fish of  claim 1 , wherein the fish is a transgenic progeny of the fish into which the exogenous sequence encoding the expression product was introduced. 
     
     
         49 . The transgenic fish of  claim 48  wherein the fish is zebrafish.