METHODS FOR STAINING CELLS FOR IDENTIFICATION AND SORTING
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 .- 20 . (canceled)
21 . A method for pre-selecting the sex of a mammalian offspring, 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 predetermined fluorescence corresponding to DNA content;
sorting the sperm based on the pre-determined fluorescence;
collecting selected sperm form the sorted sperm; and
fertilizing an egg obtained from a female animal, the female being the same species as the male animal which provided the selected sperm.
22 . The method of claim 21 , wherein the pre-determined fluorescence corresponds to a desired chromosome, chromosome fragment, an insertion or a deletion.
23 . The method of claim 21 , wherein the sperm is from a mammal.
24 . The method of claim 23 , wherein the mammal is one selected from the group consisting of bovine, swine, rabbit, alpaca, horse, dog, cat, ferret, rat, mouse and buffalo.
25 . The method of claim 21 , wherein the dye is membrane impermeant.
26 . The method of claim 25 , 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.
27 . The method of claim 21 , wherein the dye is membrane permeant.
28 . The method of claim 27 , 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.
29 . The method of claim 21 , wherein the mixing is at a temperature between about −4° C. to about 30° C.
30 . The method of claim 29 , wherein the mixing is at a temperature about 0° C., 4° C., 12° C. or 30° C.
31 . The method of claim 21 , wherein the mixing period of time is about 1 minute to about 15 minutes.
32 . The method of claim 21 , wherein the mixing period of time is less than 1 minute.
33 . The method of claim 21 , further comprising mixing the sperm with at least one nanoparticle.
34 . The method of claim 21 , wherein the nanoparticle comprises a quantum dot or metallic nanoparticle.
35 . The method of claim 21 , wherein the nanoparticle comprises a targeting molecule.
36 . The method of claim 35 , wherein the targeting molecule binds DNA or a fluorescent dye.
37 . The method of claim 21 , further comprising eliminating dead sperm before sorting the sperm.
38 . The method of claim 21 , wherein the sperm are sorted by X- or Y-chromosome DNA content with at least 90% efficiency.
39 . The method of claim 21 , wherein the viability of the sperm before sorting is at least 30%.
40 . The method of claim 39 , wherein the viability of the sperm is at least 75%.
41 . The method of claim 40 , wherein the viability of the sperm is at least 80%.
42 . The method of claim 41 , wherein the viability of the sperm is at least 90%.
43 . The method of claim 21 , wherein said fertilization is in vitro fertilization (IVF).
44 . An embryo or animal produced by the method of claim 21 .