Method for the selection of a long-term producing cell

Abstract

Herein is reported a method for determining methylation of a promoter nucleic acid operably linked to a nucleic acid encoding a polypeptide and thereby determining the long-term productivity of a cell. Also an aspect is a method for selecting a cell for producing a polypeptide by determining the methylation of the promoter nucleic acid operably linked to the structural gene encoding the polypeptide.

1 .- 13 . (canceled) 
     
     
         14 . A method for selecting a cell for the long-term production of a polypeptide comprising
 a) providing at least one cell comprising a nucleic acid comprising a structural gene encoding the polypeptide operably linked to a promoter nucleic acid,   b) identifying and determining for at least one cell the methylation of a CpG-site with high methylation frequency within the promoter nucleic acid,   c) identifying and determining for at least one cell the methylation of a cytosine at a non-CpG-site, and   d) selecting the cell for the long-term production of a polypeptide wherein the methylation determined in step b) is below a threshold value, wherein the threshold value is below twice the average value obtained by determining the methylation of a cytosine at a non-CpG-site in step c), or selecting a cell clone in which the methylation frequency as determined in step b) is below 5%.   
     
     
         15 . A method for selecting a cell clone for the long-term production of a polypeptide, said method comprising:
 a) identifying a CpG-site in a promoter nucleic acid, wherein the identifying of the CpG-site comprises:
 1) separately isolating the DNA from at least 10 cells of a cultivation of a cell that has a production rate of a polypeptide that is after a cultivation time of 30 generations of the cell in the absence of a selection agent less than 90% of the production rate of the cell after the first generation of the cultivation, 
 2) modifying the cytosine of the isolated DNA by bisulfite treatment, 
 3) identifying a CpG site within the promoter nucleic acid operably linked to the structural gene encoding the polypeptide with a methylation frequency of at least 0.2 based on the DNA obtained in step 2) and thereby identifying a CpG-site, 
   b) providing a cell clone comprising a nucleic acid comprising a structural gene encoding a polypeptide operably linked to a promoter nucleic acid which is the same as that of step a),   c) determining the methylation frequency of the CpG-site identified in step a) for a cell clone of step b), which comprises a nucleic acid comprising a structural gene encoding a polypeptide operably linked to a promoter nucleic acid which is the same as that of step a), based on at least 10 copies of the nucleic acid or 10 cells obtained from a cultivation thereof,   d) determining the methylation of a cytosine at a non-CpG-site, and   e) selecting a cell clone in which the methylation frequency as determined in step c) is below twice the average value obtained by determining the methylation of a cytosine at a non-CpG-site in step d), or selecting a cell clone in which the methylation frequency as determined in step c) is below 5%.   
     
     
         16 . The method of  claim 15 , wherein the determining in step c) comprises
 1) isolating the DNA from each of the cell clones,   2) performing for each isolated DNA individually a method selected from group consisting of: i) a methylation specific polymerase chain reaction, ii) a polymerase chain reaction with a methylation specific primer and an universal primer, and iii) individually digesting the isolated DNA with a restriction enzyme and performing a polymerase chain reaction for each of the digested DNA with a methylation specific primer and an universal primer, and   3) determining from the results obtained in step c-2) the methylation frequency of the CpG-site.   
     
     
         17 . A method for selecting a cell clone for the long-term production of a polypeptide, said method comprising:
 a) determining for each of at least one cell clone, which comprises a nucleic acid comprising a structural gene encoding a polypeptide operably linked to a promoter nucleic acid that has the nucleic acid sequence of SEQ ID NO: 01, the methylation frequency of a CpG-site at a position selected from positions 80, 96, 425, 437, 563 and 591 of SEQ ID NO: 01 based on the methylation determined for at least 10 copies of the nucleic acid or in at least 10 cells obtained from a cultivation of each cell clone,   b) selecting a cell clone in which the methylation frequency as determined is below 5%,   
       wherein the cell clone is a Chinese Hamster Ovary (CHO) cell clone. 
     
     
         18 . The method of  claim 17 , wherein the determining in step c) comprises
 1) isolating the DNA from each of the cell clones,   2) performing for each isolated DNA individually a method selected from group consisting of: i) a methylation specific polymerase chain reaction, ii) a polymerase chain reaction with a methylation specific primer and an universal primer, and iii) individually digesting the isolated DNA with a restriction enzyme and performing a polymerase chain reaction for each of the digested DNA with a methylation specific primer and an universal primer,   3) determining with the results obtained in step c-2) the methylation frequency of the CpG-site.   
     
     
         19 . The method of  claim 15 , wherein the promoter nucleic acid has the sequence of SEQ ID NO: 01 or comprises a fragment thereof or a variant thereof and the CpG-site is selected from position 80, 96, 425, 437, 563 and 591 of SEQ ID NO: 01 or a thereto corresponding position in a fragment or variant thereof. 
     
     
         20 . The method of  claim 18 , wherein the primer are independently of each other selected from the group consisting of SEQ ID NO: 06 to SEQ ID NO: 20. 
     
     
         21 . The method of  claim 20 , wherein a primer is selected from the group consisting of SEQ ID NO: 11, 14 and 15, the universal primer has the sequence of SEQ ID NO: 09 and a methylation specific primer is selected from the group consisting of SEQ ID NO: 17, 18 and 19. 
     
     
         22 . The method of  claim 21 , wherein the universal primer have the sequence of SEQ ID NO: 09 and 11 and the methylation specific primer have the sequence of SEQ ID NO: 11 and 18. 
     
     
         23 . A method for the long-term production of a polypeptide comprising:
 a) providing a mammalian, non-human cell,   b) transfecting the provided cell with a nucleic acid containing a structural gene encoding the polypeptide operably linked to a promoter nucleic acid,   c) selecting a cell clone according to the method of claim  2 ,   d) cultivating the selected cell clone, and   e) recovering the polypeptide from the cultivation medium and/or the cell clone and thereby producing a polypeptide.   
     
     
         24 . The method of  claim 23 , comprising, after step b) and prior to step c),
 i) cultivating the transfected cell clone in the presence of a selection agent,   ii) single depositing the transfected cells, and   iii) cultivating the single deposited transfected cells in the presence of a selection agent.   
     
     
         25 . A kit comprising
 a) a reagent for the modification of non-methylated cytosine in a CpG-site, and   b) a primer selected from SEQ ID NO: 13, 16, 17, 18, 19 and 20.