DNA construct, isolated molecule of nucleic acid, homologous recombinant cell and its use, method of changing expression of endogenous G-CSF gene and production process of G-CSF

摘要

In the present invention, there is described a DNA construct altering after being introduced into a mammalian cell genome through the mediation of a homologous recombination an expression of an endogenous G-CSF gene in the cell and containing: (i) a targeting sequence comprising at least 20 nucleotides following immediately each other from SEQ ID Number 5; and (ii) transcription regulatory sequence. Described is also an isolated nucleic acid molecule containing at least 100 nucleotides immediately following each other and being selected from SEQ ID Number 5 or complement thereof wherein said isolated nucleic acid molecule does not code the entire G-CSF. Further described is a homologously recombinant cell being stable transfected by a DNA construct wherein said DNA construct has been subjected to homologous recombination with a genomic DNA upstream to an initiating codon of the ATG sequence encoding an endogenous G-CSF. The invention further relates to a method of producing G-CSF, being characterized in that the aforementioned homologously recombinant cell is cultured in vitro under such conditions that enable the cell to exprime and excrete G-CSF.