Selection for more efficient transformation host cells

Abstract

This invention provides methods for producing and selecting host cells that better survive transformation treatment by subjecting host cells to conditions that alter them, subjecting the altered cells to transformation conditions, and selecting host cells that survive the transformation conditions. This invention also provides methods for transferring nucleic acids of interest into host cells, using cells that are better able to survive transformation treatment. Also, this invention provides kits for producing or selecting host cells in transformation treatments, as well as, kits comprising various host cells that may be utilized in transformation experiments.

What is claimed is:  
     
         1 . A method for producing host cells that better survive transformation treatment, comprising: 
 (a) subjecting host cells to conditions that alter at least a portion of the host cells;    (b) subjecting the host cells of (a) to transformation conditions; and    (c) selecting host cells of (b) that survive the transformation conditions.    
     
     
         2 . The method of  claim 1 , wherein the host cells are prokaryotic or eukaryotic.  
     
     
         3 . The method of  claim 2 , wherein the host cells are prokaryotic.  
     
     
         4 . The method of  claim 3 , wherein the prokaryotic cells are  E. coli.    
     
     
         5 . The method of  claim 1 , wherein the transformation conditions include electrical treatment.  
     
     
         6 . The method of  claim 5 , wherein the electrical treatment is electroporation.  
     
     
         7 . The method of  claim 6 , wherein the electroporation comprises treating the host cells to about 1.75 kV and 200 ohms.  
     
     
         8 . The method of  claim 6 , wherein the electroporation comprises treating the host cells to about 2.5 kV and 1000 ohms.  
     
     
         9 . The method of  claim 1 , wherein the transformation conditions include chemical treatment.  
     
     
         10 . The method of  claim 9 , wherein the chemical treatment is Ca 2+ -phosphate precipitation.  
     
     
         11 . The method of  claim 1 , wherein the conditions that alter the hosts cells are mutation conditions that mutate at least one of the host cells.  
     
     
         12 . The method of  claim 11 , wherein the host cells are mutated by chemical mutagens.  
     
     
         13 . The method of  claim 11 , wherein the host cells are mutated by irradiation.  
     
     
         14 . The method of  claim 13 , wherein the irradiation comprises ultraviolet irradiation.  
     
     
         15 . The method of  claim 1 , wherein the host cells transiently express Rec A protein prior to subjecting the host cells to conditions that alter them in (a).  
     
     
         16 . The method of  claim 1 , wherein the host cells are subjected at least twice to conditions that alter them.  
     
     
         17 . The method of  claim 1 , wherein the host cells are subjected at least twice to transformation conditions.  
     
     
         18 . The method of  claim 1 , further comprising culturing the selected host cells of (c) in a selected media capable of promoting their growth.  
     
     
         19 . A method for selecting host cells that better survive transformation treatment, comprising: 
 (a) obtaining host cells that have been altered;    (b) subjecting the host cells of (a) to transformation conditions; and    (c) selecting host cells of (b) that survive the transformation conditions.    
     
     
         20 . The method of  claim 19 , wherein the host cells are prokaryotic or eukaryotic.  
     
     
         21 . The method of  claim 20 , wherein the prokaryotic cells are  E. coli.    
     
     
         22 . The method of  claim 19 , wherein the transformation conditions include electrical treatment.  
     
     
         23 . The method of  claim 22 , wherein electrical treatment is electroporation.  
     
     
         24 . The method of  claim 23 , wherein the electroporation comprises treating the host cells to about 1.75 kV and 200 ohms.  
     
     
         25 . The method of  claim 23 , wherein the electroporation comprises treating the host cells to about 2.5 kV and 1000 ohms.  
     
     
         26 . The method of  claim 19 , wherein the transformation conditions include chemical treatment.  
     
     
         27 . The method of  claim 26 , wherein the chemical treatment comprises Ca 2+ -phosphate precipitation.  
     
     
         28 . The method of  claim 19 , wherein the conditions that alter the hosts cells are mutation conditions that mutate at least one of the host cells.  
     
     
         29 . The method of  claim 28 , wherein the host cells are mutated by chemical mutagens.  
     
     
         30 . The method of  claim 28 , wherein the host cells are mutated by irradiation.  
     
     
         31 . The method of  claim 30 , wherein the irradiation comprises ultraviolet irradiation.  
     
     
         32 . The method of  claim 19 , wherein the host cells transiently express Rec A protein prior to subjecting the host cells to conditions that alter them in (a).  
     
     
         33 . The method of  claim 19 , wherein the host cells are subjected at least twice to conditions that alter them.  
     
     
         34 . The method of  claim 19 , wherein the host cells are subject at least twice to transformation conditions.  
     
     
         35 . The method of  claim 19 , further comprising culturing the selected host cells of (c) in a selected media capable of promoting their growth.  
     
     
         36 . Host cells produced by the method of  claim 1 .  
     
     
         37 . The host cells of  claim 36 , wherein the host cells are prokaryotic or eukaryotic.  
     
     
         38 . The host cells of  claim 37 , wherein the prokaryotic cells are  E. coli.    
     
     
         39 . Host cells selected by the method of  claim 19 .  
     
     
         40 . The host cells of  claim 39 , wherein the host cells are prokaryotic or eukaryotic.  
     
     
         41 . The host cells of  claim 40 , wherein the prokaryotic cells are  E. coli.    
     
     
         42 . A bacterial strain designated XL1-Blue MRF′ B150.  
     
     
         43 . A method for producing host cells that better survive transformation treatment, comprising: 
 (a) subjecting host cells to transformation conditions; and    (b) selecting host cells of (a) that survive the transformation conditions.    
     
     
         44 . The method of  claim 43 , wherein the host cells are cells in which a spontaneous mutation has occurred.  
     
     
         45 . A method for transferring nucleic acids of interest into host cells, comprising: 
 (a) combining host cells produced by the method of  claim 1  with nucleic acids of interest;    (b) subjecting the host cells to transformation conditions; and    (c) selecting host cells of (b) that survive the transformation conditions and that contain the nucleic acids of interest.    
     
     
         46 . The method of  45 , further comprising culturing the cells transformed in (c) in a selected media capable of promoting their growth.  
     
     
         47 . The method of  45 , wherein host cells are prokaryotic and eukaryotic.  
     
     
         48 . The method  claim 47 , wherein the prokaryotic cells are  E. coli.    
     
     
         49 . The method of  claim 45 , wherein the transformation conditions include electrical treatment.  
     
     
         50 . The method of  claim 49 , wherein the electrical treatment is electroporation.  
     
     
         51 . The method of  claim 45 , wherein the transformation conditions include chemical treatment.  
     
     
         52 . The method of  claim 51 , wherein the chemical treatment is Ca 2+ -phosphate precipitation.  
     
     
         53 . The method of  claim 45 , wherein the nucleic acids of interest are molecular beacons.  
     
     
         54 . The method of  claim 45 , wherein the nucleic acids of interest are peptide nucleic acids.  
     
     
         55 . The method of  claim 45 , wherein the nucleic acids of interest are single-stranded RNA.  
     
     
         56 . The method of  claim 45 , wherein the nucleic acids of interest are double-stranded RNA.  
     
     
         57 . The method of  claim 45 , wherein the nucleic acids of interest are modified nucleic acids.  
     
     
         58 . A method for transferring nucleic acids of interest into host cells, comprising: 
 (a) combining host cells selected by the method of  claim 19  with nucleic acids of interest;    (b) subjecting the host cells to transformation conditions; and    (c) selecting host cells of (b) that survive the transformation conditions and that contain the nucleic acids of interest.    
     
     
         59 . The method of  58 , further comprising culturing the transformed cells in a selected media capable of promoting their growth.  
     
     
         60 . The method of  58 , wherein host cells are prokaryotic and eukaryotic.  
     
     
         61 . The method  claim 60 , wherein the prokaryotic cells are  E. coli.    
     
     
         62 . The method of  claim 58 , wherein the transformation conditions include electrical treatment.  
     
     
         63 . The method of  claim 62 , wherein the electrical treatment is electroporation.  
     
     
         64 . The method of  claim 58 , wherein the transformation conditions include chemical treatment.  
     
     
         65 . The method of  claim 64 , wherein the chemical treatment is Ca 2+ -phosphate precipitation.  
     
     
         66 . The method of  claim 58 , wherein the nucleic acids of interest are molecular beacons.  
     
     
         67 . The method of  claim 58 , wherein the nucleic acids of interest are peptide nucleic acids.  
     
     
         68 . The method of  claim 58 , wherein the nucleic acids of interest are nucleic acids that are modified by a chemical covalent linkage.  
     
     
         69 . A method for transferring nucleic acids of interest into host cells, comprising: 
 (a) combining host cells produced by the method of  claim 43  with nucleic acids of interest;    (b) subjecting the host cells to transformation conditions; and    (c) selecting host cells of (b) that survive the transformation conditions and that contain the nucleic acids of interest.    
     
     
         70 . A kit used in the practice of the method of  claim 1 .  
     
     
         71 . A kit used in the practice of the method of  claim 19 .  
     
     
         72 . A kit used in the practice of the method of  claim 45 .  
     
     
         73 . A kit used in the practice of the method of  claim 58 .  
     
     
         74 . A kit comprising the host cells of  claim 36 .  
     
     
         75 . A kit comprising the host cells of  claim 39 .  
     
     
         76 . A kit comprising the bacterial strain of  claim 42.