Genomic sequences for protein production and delivery

Abstract

An isolated nucleic acid molecule that hybridizes under stringent conditions, or shares at least 80% sequence identity, with a defined genomic region upstream of the coding region of a FSHβ gene, and a DNA construct containing that nucleic acid molecule as a targeting sequence for homologous recombination.

What is claimed is:  
     
         1 . A DNA construct that alters expression of an endogenous FSHβ gene in a mammalian cell upon integration into the genome of the cell via homologous recombination, the construct comprising a targeting sequence containing at least 20 contiguous nucleotides from SEQ ID NO:4 or 5 and a transcriptional regulatory sequence.  
     
     
         2 . The DNA construct of    claim 1   , wherein the construct further comprises an exon and a splice-donor site.  
     
     
         3 . The DNA construct of    claim 2   , wherein the construct further comprises, downstream from the splice-donor site, an intron and a splice-acceptor site.  
     
     
         4 . The DNA construct of    claim 1   , wherein the construct further comprises a selectable marker gene.  
     
     
         5 . The DNA construct of    claim 1   , wherein the target sequence contains at least 50 contiguous nucleotides from SEQ ID NO:4 or 5.  
     
     
         6 . An isolated nucleic acid comprising at least 20 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         7 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises at least 50 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         8 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises at least 100 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         9 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises at least 200 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         10 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises at least 500 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         11 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises at least 1000 contiguous nucleotides of SEQ ID NO:4 or its complement.  
     
     
         12 . The isolated nucleic acid of    claim 6   , wherein the isolated nucleic acid comprises SEQ ID NO:4 or its complement.  
     
     
         13 . An isolated nucleic acid comprising at least 20 contiguous nucleotides of SEQ ID NO:5 or its complement.  
     
     
         14 . The isolated nucleic acid of    claim 13   , wherein the isolated nucleic acid comprises at least 50 contiguous nucleotides of SEQ ID NO:5 or its complement.  
     
     
         15 . The isolated nucleic acid of    claim 13   , wherein the isolated nucleic acid comprises at least 100 contiguous nucleotides of SEQ ID NO:5 or its complement.  
     
     
         16 . The isolated nucleic acid of    claim 13   , wherein the isolated nucleic acid comprises at least 200 contiguous nucleotides of SEQ ID NO:5 or its complement.  
     
     
         17 . The isolated nucleic acid of    claim 13   , wherein the isolated nucleic acid comprises SEQ ID NO:5 or its complement.  
     
     
         18 . An isolated DNA comprising a strand that comprises a nucleotide sequence that (i) is at least 100 nucleotides in length and (ii) hybridizes under highly stringent conditions with SEQ ID NO:4 or 5, or the complement of SEQ ID NO:4 or 5.  
     
     
         19 . The isolated DNA of    claim 18   , wherein the nucleotide sequence is at least 200 nucleotides in length.  
     
     
         20 . The isolated DNA of    claim 18   , wherein the nucleotide sequence is at least 400 nucleotides in length.  
     
     
         21 . The isolated DNA of    claim 18   , wherein the nucleotide sequence is at least 1,000 nucleotides in length.  
     
     
         22 . An isolated DNA comprising a strand that comprises a nucleotide sequence that (i) is at least 100 nucleotides in length and (ii) shares at least 80% sequence identity with a fragment of SEQ ID NO:4 or 5 having the same length as the nucleotide sequence.  
     
     
         23 . The isolated DNA of    claim 22   , wherein the nucleotide sequence is at least 200 nucleotides in length.  
     
     
         24 . The isolated DNA of    claim 22   , wherein the nucleotide sequence is at least 400 nucleotides in length.  
     
     
         25 . The isolated DNA of    claim 22   , wherein the nucleotide sequence is at least 1,000 nucleotides in length.  
     
     
         26 . A homologously recombinant cell stably transfected with the DNA construct of    claim 1   , the DNA construct having undergone homologous recombination with genomic DNA upstream of the ATG initiation codon of an endogenous FSHβ coding sequence.  
     
     
         27 . A homologously recombinant cell stably transfected with the DNA construct of    claim 2   , the DNA construct having undergone homologous recombination with genomic DNA upstream of the ATG initiation codon of an endogenous FSHβ coding sequence.  
     
     
         28 . A homologously recombinant cell stably transfected with the DNA construct of    claim 3   , the DNA construct having undergone homologous recombination with genomic DNA upstream of the ATG initiation codon of an endogenous FSHβ coding sequence.  
     
     
         29 . A homologously recombinant cell stably transfected with the DNA construct of    claim 4   , the DNA construct having undergone homologous recombination with genomic DNA upstream of the ATG initiation codon of an endogenous FSHβ coding sequence.  
     
     
         30 . A method of altering expression of an endogenous FSHβ gene in a mammalian cell, the method comprising 
 introducing the DNA construct of    claim 1    into the cell;  
 maintaining the cell under conditions which permit homologous recombination to occur between the construct and a genomic target site homologous to the targeting sequence, to produce a homologously recombinant cell; and  
 maintaining the homologously recombinant cell under conditions which permit expression of the FSHβ coding sequence under the control of the transcriptional regulatory sequence.  
 
     
     
         31 . A method of delivering FSHβ to an animal, comprising 
 providing the cell of    claim 26   , and  
 implanting the cell in the animal, wherein the cell secretes FSHβ.  
 
     
     
         32 . A method of delivering FSHβ to an animal, comprising 
 providing the cell of    claim 27   , and  
 implanting the cell in the animal, wherein the cell secretes FSHβ.  
 
     
     
         33 . A method of delivering FSHβ to an animal, comprising 
 providing the cell of    claim 28   , and  
 implanting the cell in the animal, wherein the cell secretes FSHβ.  
 
     
     
         34 . A method of delivering FSHβ to an animal, comprising 
 providing the cell of    claim 29   , and  
 implanting the cell in the animal, wherein the cell secretes FSHβ.  
 
     
     
         35 . A method of producing FSHβ comprising 
 providing the cell of    claim 26   , and  
 culturing the cell in vitro under conditions which permit the cell to express and secrete FSHβ.  
 
     
     
         36 . A method of producing FSH=, comprising 
 providing the cell of    claim 27   , and    culturing the cell in vitro under conditions which permit the cell to express and secrete FSHβ.    
     
     
         37 . A method of producing FSHβ, comprising 
 providing the cell of    claim 28   , and  
 culturing the cell in vitro under conditions which permit the cell to express and secrete FSHβ.  
 
     
     
         38 . A method of producing FSHβ, comprising 
 providing the cell of    claim 29   , and  
 culturing the cell in vitro under conditions which permit the cell to express and secrete FSHβ.