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 the G-CSF gene, and a DNA construct containing that DNA molecule as a targeting sequence for homologous recombination.

What is claimed is:  
     
         1 . A DNA construct that alters expression of an endogenous G-CSF gene in a mammalian cell upon integration into the genome of the cell via homologous recombination, the construct comprising: (i) a targeting sequence containing at least 20 contiguous nucleotides from SEQ ID NO:5 and (ii) 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 targeting sequence contains at least 50 contiguous nucleotides from SEQ ID NO:5.  
     
     
         6 . An isolated nucleic acid comprising at least 20 contiguous nucleotides of SEQ ID NO:5 or its complement, wherein the isolated nucleic acid does not encode full-length G-CSF.  
     
     
         7 . The isolated nucleic acid of  claim 6 , wherein the isolated nucleic acid comprises at least 50 contiguous nucleotides of SEQ ID NO:5 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:5 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:5 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:5 or its complement.  
     
     
         11 . The isolated DNA of  claim 6 , wherein the isolated nucleic acid comprises nucleotides 1470 to 4723 of SEQ ID NO:5, or its complement.  
     
     
         12 . The isolated DNA of  claim 6 , wherein the isolated nucleic acid comprises SEQ ID NO:5 or its complement.  
     
     
         13 . An isolated nucleic acid 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:5 or the complement thereof.  
     
     
         14 . The isolated nucleic acid of  claim 13 , wherein the nucleotide sequence is at least 200 nucleotides in length.  
     
     
         15 . The isolated nucleic acid of  claim 13 , wherein the nucleotide sequence is at least 400 nucleotides in length.  
     
     
         16 . The isolated nucleic acid of  claim 13 , wherein the nucleotide sequence is at least 1,000 nucleotides in length.  
     
     
         17 . An isolated nucleic acid 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:5 having the same length as the nucleotide sequence.  
     
     
         18 . The isolated nucleic acid of  claim 17 , wherein the nucleotide sequence is at least 200 nucleotides in length.  
     
     
         19 . The isolated nucleic acid of  claim 18 , wherein the nucleotide sequence is at least 400 nucleotides in length.  
     
     
         20 . The isolated nucleic acid of  claim 18 , wherein the nucleotide sequence is at least 1,000 nucleotides in length.  
     
     
         21 . 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 G-CSF coding sequence.  
     
     
         22 . 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 G-CSF coding sequence.  
     
     
         23 . 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 G-CSF coding sequence.  
     
     
         24 . 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 G-CSF coding sequence.  
     
     
         25 . A method of altering expression of an endogenous G-CSF 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 G-CSF coding sequence under the control of the transcriptional regulatory sequence.    
     
     
         26 . A method of altering expression of an endogenous G-CSF gene in a mammalian cell, the method comprising 
 introducing the DNA construct of  claim 4  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 G-CSF coding sequence under the control of the transcriptional regulatory sequence.    
     
     
         27 . A method of delivering G-CSF to an animal, comprising 
 providing the cell of  claim 21 , and    implanting the cell in the animal, wherein the cell secretes G-CSF.    
     
     
         28 . A method of delivering G-CSF to an animal, comprising 
 providing the cell of  claim 22 , and    implanting the cell in the animal, wherein the cell secretes G-CSF.    
     
     
         29 . A method of delivering G-CSF to an animal, comprising 
 providing the cell of  claim 23 , and    implanting the cell in the animal, wherein the cell secretes G-CSF.    
     
     
         30 . A method of delivering G-CSF to an animal, comprising 
 providing the cell of  claim 24 , and    implanting the cell in the animal, wherein the cell secretes G-CSF.    
     
     
         31 . A method of producing G-CSF, comprising 
 providing the cell of  claim 21 , and    culturing the cell in vitro under conditions which permit the cell to express and secrete G-CSF.    
     
     
         32 . A method of producing G-CSF, comprising 
 providing the cell of  claim 22 , and    culturing the cell in vitro under conditions which permit the cell to express and secrete G-CSF.    
     
     
         33 . A method of producing G-CSF, comprising 
 providing the cell of  claim 23 , and    culturing the cell in vitro under conditions which permit the cell to express and secrete G-CSF.    
     
     
         34 . A method of producing G-CSF, comprising 
 providing the cell of  claim 24 , and    culturing the cell in vitro under conditions which permit the cell to express and secrete G-CSF.