Methods for staining cells for identification and sorting

Abstract

The present invention provides novel methods of cell staining, such as bovine sperm, using electroporation or osmolality treatments at viability-enhancing temperatures. Furthermore, methods of highly efficient cell sorting that are especially suitable in sorting bovine sperm using novel cell staining procedures are also provided.

1 . A method for staining sperm, comprising: 
 staining sperm with a dye by mixing the sperm with the dye; and    electroporating the sperm and dye for a period of time to provide substantially uniform staining and concomitantly to substantially preserve sperm viability.    
     
     
         2 . A method for distinguishing sperm based on DNA content, comprising: 
 staining sperm with a DNA-selective fluorescent dye by mixing the sperm with the dye at a temperature substantially equal to or less than 39° C.;    electroporating the sperm and dye for a period of time to provide substantially uniform staining and concomitantly to substantially preserve sperm viability;    exposing the sperm to a light source to cause the stained DNA to fluoresce;    detecting a pre-determined fluorescence of the stained DNA, the pre-determined fluorescence corresponding to DNA content;    sorting the sperm based on the pre-determined fluorescence; and    collecting selected sperm form the sorted sperm.    
     
     
         3 . The method of  claim 2 , wherein the pre-determined fluorescence corresponds to a desired chromosome, chromosome fragment, an insertion or a deletion.  
     
     
         4 . The method of  claim 2 , wherein the sperm is from a mammal.  
     
     
         5 . The method of  claim 4 , wherein the mammal is one selected from the group consisting of bovine, swine, rabbit, alpaca, horse, dog, cat, ferret, rat, mouse and buffalo.  
     
     
         6 . The method of  claim 2 , wherein the dye is membrane impermeant.  
     
     
         7 . The method of  claim 6 , wherein the dye comprises at least one selected from the group consisting of SYTOX blue, SYTOX green, SYTOX orange, a cyanine dimer, POPO-1, BOBO-1, YOYO-1, TOTO-1, JOJO-1, POPO-3, LOLO-1, BOBO-3, YOYO-3, TOTO-3, a cyanine monomer, PO-PRO-1, BO-PRO-1, YO-PRO-1, TO-PRO-1, JO-PRO-1, PO-PRO-3, LO-PRO-1, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5, acridine homodimer, 7-amino actinomycin D, ethidium bromide, ethidium homodimer-1, ethidium homodimer-2, ethidium nonazide, nuclear yellow and propidium iodide.  
     
     
         8 . The method of  claim 2 , wherein the dye is membrane permeant.  
     
     
         9 . The method of  claim 8 , wherein the dye comprises at least one selected from the group consisting of SYTO 40 blue-fluorescent nucleic acid stain, SYTO 41 blue, SYTO 42 blue, SYTO 43 blue, SYTO 44 blue, SYTO 45 blue, a green-fluorescent SYTO dye, SYTO 9 green, SYTO 10 green, SYTO BC green, SYTO 13 green, SYTO 16 green, SYTO 24 green, SYTO 21 green, SYTO 27 green, SYTO 26 green, SYTO 23 green, SYTO 12 green, SYTO 11 green, SYTO 20 green, SYTO 22 green, SYTO 15 green, SYTO 14 green, SYTO 25 green, an orange-fluorescent SYTO dye, SYTO 86 orange, SYTO 81 orange, SYTO 80 orange, SYTO 82 orange, SYTO 83 orange, SYTO 84 orange, SYTO 85 orange, a red-fluorescent SYTO dye, SYTO 64 red, SYTO 61 red, SYTO 17 red, SYTO 59 red, SYTO 62 red, SYTO 60 red, SYTO 63 red, a Hoechst dye, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, LDS 751 and dihydroethidium.  
     
     
         10 . The method of  claim 2 , wherein the mixing is at a temperature between about −4° C. to about 30° C.  
     
     
         11 . The method of  claim 2 , wherein the mixing is at a temperature about 0° C., 4° C., 12° C. or 30° C.  
     
     
         12 . The method of  claim 2 , wherein the mixing period of time is about 1 minute to about 15 minutes.  
     
     
         13 . The method of  claim 2 , wherein the mixing period of time is less than 1 minute.  
     
     
         14 . The method of  claim 2 , further comprising mixing the sperm with at least one nanoparticle.  
     
     
         15 . The method of  claim 2 , wherein the nanoparticle comprises a quantum dot or metallic nanoparticle.  
     
     
         16 . The method of  claim 2 , wherein the nanoparticle comprises a targeting molecule.  
     
     
         17 . The method of  claim 2 , wherein the targeting molecule binds DNA or a fluorescent dye.  
     
     
         18 . The method of  claim 2 , further comprising eliminating dead sperm before sorting the sperm.  
     
     
         19 . The method of  claim 2 , wherein the sperm are sorted by X- or Y-chromosome DNA content with at least 90% efficiency.  
     
     
         20 . The method of  claim 2 , wherein the viability of the sperm before sorting is at least 30%.  
     
     
         21 . The method of  claim 20 , wherein the viability of the sperm is at least 75%.  
     
     
         22 . The method of  claim 21 , wherein the viability of the sperm is at least 80%.  
     
     
         23 . The method of  claim 22 , wherein the viability of the sperm is at least 90%.  
     
     
         24 . A method for distinguishing sperm based on DNA content and maintaining sperm viaiblity, comprising: 
 staining sperm with a DNA-selective fluorescent dye by mixing the sperm with the dye at a temperature substantially sufficient to maintain a comparatively high sperm viability rate;    electroporating the sperm and dye for a period of time to provide substantially uniform staining and concomitantly to substantially preserve sperm viability;    exposing the sperm to a light source to cause the stained DNA to fluoresce;    detecting a predetermined fluorescence of the stained DNA, the pre-determined fluorescence corresponding to DNA content;    sorting the sperm based on the pre-determined fluorescence; and    collecting selected sperm from the sorted sperm.    
     
     
         25 . The method of  claim 24 , wherein the high sperm viability rate is at least 70%.  
     
     
         26 . A method for distinguishing sperm based on DNA content, wherein the sperm are stained with a DNA-selective fluorescent dye; comprising: 
 incubating the sperm under a hypertonic condition and at a temperature substantially sufficient to maintain a comparatively high sperm viability rate;    transferring the sperm to a hypotonic condition;    exposing the sperm to a light source to cause the stained DNA to fluoresce;    detecting a pre-determined fluorescence of the stained DNA, the pre-determined fluorescence corresponding to DNA content;    sorting the sperm based on the pre-determined fluorescence; and    collecting selected sperm from the sorted sperm,    wherein the dye is present in a least one of the hypertonic or hypotonic condition.    
     
     
         27 . The method of  claim 26 , wherein the hypertonic condition is about less than 250 mOsm and the hypotonic conditions is about greater than 250 mOsm.  
     
     
         28 . The method of  claim 27 , wherein the incubation temperature is less than or equal to 4° C.  
     
     
         29 . A method for distinguishing sperm based on DNA content while maintaining sperm viability, comprising: 
 incubating the sperm under a hypertonic condition to partially dehydrate the sperm, and at a temperature substantially sufficient to maintain a comparatively high sperm viability rate;    transferring the sperm to a hypotonic condition;    exposing the sperm to a light source to cause the stained DNA to fluoresce;    detecting a pre-determined fluorescence of the stained DNA, the pre-determined fluorescence corresponding to DNA content;    sorting the sperm based on the pre-determined fluorescence; and    collecting selected sperm from the sorted sperm,    wherein the dye is present in a least one of the hypertonic or hypotonic condition.    
     
     
         30 . The method of  claim 29 , wherein the high sperm viability rate is greater than 70%.  
     
     
         31 . A method to pre-select the sex of mammalian offspring comprising: 
 sorting sperm according to the method of  claim 2;  and    fertilizing an egg obtained from a female animal, the female being the same species as the male animal which provided the selected sperm.    
     
     
         32 . A method to pre-select the sex of a mammalian offspring comprising: 
 sorting sperm according to the method of  claim 2;  and    inseminating a female animal, the female being the same species as the male animal which provided the selected sperm.    
     
     
         33 . A method for staining sperm, comprising permeating the sperm cell membrane.