Claims for Patent: 6,380,458

Summary for Patent: 6,380,458

Title:	Cell-lineage specific expression in transgenic zebrafish
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.
Inventor(s):	Lin; Shuo (Augusta, GA)
Assignee:	Medical College of Georgia Research Institute, Inc. (Augusta, GA)
Application Number:	08/871,755
Patent Claims:	1. A transgenic zebrafish that expresses a heterologous expression product, comprising a zebrafish cell lineage-specific expression sequence operably linked to a sequence encoding a heterologous expression product, wherein the sequence encoding the expression product is integrated into the genome of the zebrafish and wherein expression of the heterologous expression product is cell lineage-specific. 2. The transgenic zebrafish of claim 1 wherein the expression product is a reporter protein. 3. The transgenic zebrafish of claim 2 wherein the reporter protein is selected from the group consisting of .beta.-galactosidase, chloramphenicol acetyltransferase, and green fluorescent protein. 4. The transgenic zebrafish of claim 3 wherein the reporter protein is green fluorescent protein. 5. The transgenic zebrafish 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. 6. The transgenic zebrafish of claim 5 wherein the expression product is expressed only in blood cells. 7. The transgenic zebrafish of claim 6 wherein the expression product is expressed only in erythroid progenitor cells. 8. The transgenic zebrafish of claim 5 wherein the expression product is expressed only in neurons. 9. The transgenic zebrafish of claim 1 wherein expression of the expression product is stable and transmitted through the germline. 10. The transgenic zebrafish of claim 1, wherein the zebrafish expression sequence and the sequence encoding the expression product are contained in an exogenous construct. 11. The transgenic zebrafish of claim 10 wherein the expression sequence is selected from the group consisting of a GATA-1 expression sequence and a GATA-2 expression sequence. 12. The transgenic zebrafish of claim 11 wherein the expression sequence comprises a GATA-1 expression sequence. 13. The transgenic zebrafish of claim 11 wherein the expression sequence comprises a GATA-2 expression sequence. 14. The transgenic zebrafish of claim 13, wherein the expression sequence comprises the GATA-2 promoter operably linked to the neuron-specific enhancer of GATA-2. 15. The transgenic zebrafish of claim 13 wherein the expression sequence comprises the GATA-2 promoter operably linked to the blood-specific enhancer of GATA-2. 16. The transgenic zebrafish of claim 13 wherein the expression sequence comprises the GATA-2 promoter operably linked to the skin-specific enhancer of GATA-2. 17. The transgenic zebrafish of claim 10 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. 18. The transgenic zebrafish of claim 1 wherein the expression sequence is a promoter. 19. The transgenic zebrafish of claim 1 wherein the transgenic zebrafish is developed from, or is the progeny of a transgenic zebrafish developed from, an embryonic cell into which the sequence encoding the heterologous expression product was introduced. 20. The transgenic zebrafish of claim 1 wherein the expression product is expressed in predetermined cell lineages. 21. The transgenic zebrafish of claim 1 wherein the sequence encoding the heterologous expression product is genetically linked to an identified mutant gene. 22. The transgenic zebrafish of claim 1 wherein the expression sequence comprises a zebrafish promoter operably linked to a zebrafish enhancer. 23. The transgenic zebrafish of claim 22 wherein the expression sequence further comprises zebrafish 5' untranslated sequences operably linked to the promoter and the sequence encoding the expression product. 24. Cells isolated from the transgenic zebrafish of claim 1 wherein the cells express the expression product. 25. A method of making transgenic zebrafish, the method comprising: (a) introducing an exogenous construct into a zebrafish egg cell or embryonic cell, wherein the construct comprises a zebrafish 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 zebrafish, wherein expression of the expression product in the zebrafish is cell lineage-specific, wherein the exogenous construct is integrated into the genome of the zebrafish. 26. The method of claim 25 wherein the expression product is expressed in predetermined cell lineages. 27. The method of claim 25 wherein the method further comprises producing progeny of the zebrafish. 28. The method of claim 25 wherein the expression sequence comprises a zebrafish promoter operably linked to a zebrafish enhancer. 29. A method of identifying a compound that affects cell lineage-specific expression, the method comprising exposing the zebrafish of claim 1 or progeny of the zebrafish to a test compound, detecting the expression product in the zebrafish exposed to the test compound, and comparing the pattern of expression of the expression product in the zebrafish exposed to the test compound with the pattern of expression of the expression product in the zebrafish of claim 1 or progeny of the zebrafish not exposed to the test compound, wherein if the pattern of expression of the expression product in the zebrafish exposed to the test compound differs from the pattern of expression in the zebrafish not expo ed to the test compound, then the test compound affects cell-lineage specific expression. 30. A method of identifying the pattern of expression of a zebrafish gene, the method comprising detecting the expression product in the zebrafish of claim 1 or progeny of the zebrafish, wherein the expression sequence is an expression sequence of a zebrafish gene, wherein the pattern of expression of the expression product in the zebrafish or progeny of the zebrafish identifies the pattern of expression of the zebrafish gene. 31. A method of identifying a mutant gene that affects expression of a zebrafish gene, the method comprising crossing the zebrafish of claim 10 or progeny of the zebrafish to a second zebrafish having an identified mutant gene to produce a third zebrafish having both the exogenous construct and the identified mutation, wherein the expression sequence is an expression sequence of a zebrafish gene, detecting the expression product in the third zebrafish or progeny of the third zebrafish, and comparing the pattern of expression of the expression product in the third zebrafish or the progeny of the third zebrafish with the pattern of expression of the expression product in the zebrafish, wherein if the pattern of expression of the expression product in the third zebrafish or progeny of the third zebrafish differs from the pattern of expression in the zebrafish, then the mutant gene affects expression of the zebrafish gene. 32. A method of marking a mutant gene, the method comprising crossing the zebrafish of claim 10 or progeny of the zebrafish to a second zebrafish 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 zebrafish having both the exogenous construct and the mutant gene, and crossing the third zebrafish to a fourth zebrafish, wherein the fourth zebrafish has neither the exogenous construct nor the mutant gene, to produce a fifth zebrafish, wherein the fifth zebrafish has both the exogenous construct and the mutant gene, wherein the mutant gene is marked by the exogenous construct in the fifth zebrafish. 33. The method of claim 32, wherein the method further comprises crossing the fifth zebrafish, or a progeny of the fifth zebrafish, with a sixth zebrafish, and identifying progeny zebrafish expressing the expression product, wherein zebrafish expressing the expression product have the mutant gene. 34. A method of identifying genes, the method comprising: isolating from the zebrafish of claim 1 or progeny thereof a cell expressing a cell-lineage specific expression product, and identifying genes that are expressed in the cell. 35. The method of claim 34, wherein the gene is identified by extracting RNA and performing differential display. 36. The method of claim 34, wherein the gene is identified by extracting RNA and performing subtractive hybridization. 37. A method for identifying genes involved in cell lineage specific expression comprising: a. introducing a mutation into the zebrafish of claim 1 and b. detecting a change in the expression product in the fish containing the mutation, whereby a change in the expression product identifies a gene involved in cell lineage specific expression. 38. A method of identifying compounds that affect expression of genes comprising: a) contacting a zebrafish of claim 1 wherein the expression product is a directly detectable reporter protein with a compound; b) comparing the expression of the expression product in zebrafish contacted with the compound with zebrafish that were not contacted with the compound; c) determining the effect of the compound on the expression product.

Details for Patent 6,380,458

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26