Gender-specific separation of sperm cells and embryos

• US 9,879,222 B2
• Filed: 12/15/2008
• Issued: 01/30/2018
• Est. Priority Date: 12/14/2007
• Status: Active Grant

• Current Assignee: Mofa Group, Llc
• Original Assignee: Mofa Group, Llc
• Inventors: Didion, Bradley, Erwin, Whitney, Bleher, Reiner
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US12/747,988
• Publication Number:
  US 20100311059A1
• Time in Patent Office: 3,333 Days
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/6879 for sex determination

Abstract

Disclosed are sperm cells or embryos comprising a labeled oligonucleotide bound to a gender-specific repeat sequence. Methods for separating sperm cells or embryos containing a labeled oligonucleotide from sperm cells not containing the labeled oligonucleotide produce gender-enriched sperm cell fractions. The separated fractions are useful in producing offspring of a predetermined sex.

Claims

1. A method for separating a population of sperm cells, the method comprising:

a) contacting the population of sperm cells with a labeled oligonucleotide moiety capable of binding a gender-specific tandem repeat sequence on either the Y chromosome or the X chromosome in a portion of the population of sperm cells to provide a labeled fraction and an unlabeled fraction, wherein the gender-specific tandem repeat sequence contains a target sequence having consecutive purine sequences or consecutive homopyrimidine sequences, wherein the target sequence of the gender-specific tandem repeat sequence has about 12 to about 24 nucleotides, wherein the gender-specific tandem repeat sequence is repeated at least 50 times on the respective chromosome of the labeled fraction, wherein the labeled oligonucleotide moiety comprises a triplex forming oligonucleotide moiety that binds to the target sequence, and wherein the labeled oligonucleotides moiety comprises a CY3 dye linked to a 5′

-end of the triplex forming oligonucleotide with an ester bond;

b) detecting a signal that distinguishes the labeled fraction and the unlabeled fraction; and

c) separating the labeled fraction from the unlabeled fraction forming a separated labeled fraction and a separated unlabeled fraction, wherein at least about 40% of each of the separated labeled fraction and the separated unlabeled fractions contain viable sperm cells that can be used for in vivo or in vitro fertilization.

2. The method of claim 1, wherein the oligonucleotide moiety comprises a purine triplex forming oligonucleotide, a pyrimidine triplex forming oligoneucleotide, or an OligoTRIP.

3. The method of claim 1, wherein a plurality of oligonucleotide moieties bind to the gender-specific tandem repeat sequence in step (a).

4. The method of claim 1, wherein the oligonucleotide moiety further comprises a detectable label chosen from a fluorescent tag, a heavy density tag, a magnetic tag, a nanoparticle, and combinations thereof.

5. The method of claim 1, wherein the gender-specific tandem repeat sequence comprises a polynucleotide having at least 80% identity to SEQ ID NO:

1, a polynucleotide having at least 80% identity to SEQ ID NO;

2, a polynucleotide having at least 80% identity to SEQ ID NO;

3, or a polynucleotide having at least 80% identity to SEQ ID NO;

4.

6. The method of claim 5, wherein the oligonucleotide moiety comprises sequence C (SEQ ID NO:

14) or sequence D (SEQ ID NO;

15).

7. The method of claim 5, wherein the gender-specific sequence comprises SEQ ID NO:

2 or SEQ ID NO;

3.

8. The method of claim 5, wherein at least 70% of the cells of the labeled fraction comprise a Y chromosome and at least 70% of the cells of the unlabeled fraction comprise a X chromosome.

9. The method of claim 5, wherein at least 70% of cells of the labeled fraction comprise an X chromosome and at least 70% of the cells of the unlabeled fraction comprise a Y chromosome.

10. The method of claim 1, wherein at least 50% of the cells of the labeled fraction or the unlabeled fraction are viable after step (c).

11. The method of claim 1, wherein separating the cells in step (c) comprises separation by flow cytometry, centrifugation, or magnetic force.

12. The method of claim 1, wherein the gender-specific tandem repeat sequence comprises a telomeric sequence.

13. The method of claim 1, wherein the gender-specific tandem repeat sequence is from about 2,000 to about 10,000 nucleotides.

14. The method of claim 1, wherein the labeled oligonucleotide is from about 12 to about 24 nucleotides.

15. The method of claim 1, wherein the sperm cells comprise mammalian sperm cells selected from bovine, porcine, canine, and equine sperm cells.

16. A method for separating a population of sperm cells having an X chromosome and a Y chromosome, the method comprising:

a) contacting the population of sperm cells with a labeled oligonucleotide moiety capable of binding to a gender-specific tandem repeat sequence that is repeated on either the Y chromosome or the X chromosome in a portion of the population of sperm cells to provide a labeled fraction and an unlabeled fraction, wherein the gender-specific tandem repeat sequence contains a target sequence having consecutive purine sequences or consecutive homopyrimidine sequences, wherein the target sequence of the gender-specific tandem repeat sequence has about 12 to about 24 nucleotides, wherein the labeled oligonucleotide moiety is capable of binding to a major groove of a DNA duplex of the gender-specific tandem repeat sequence in the labeled fraction, and wherein the gender-specific tandem repeat sequence is repeated at least 50 times on the respective chromosome of the labeled fraction, wherein the labeled oligonucleotide moiety comprises a triplex forming oligonucleotide moiety that binds to the target sequence, and wherein the labeled oligonucleotides moiety comprises a CY3 dye linked to a 5′

-end of the triplex forming oligonucleotide with an ester linkage;

b) detecting a signal that distinguishes the labeled fraction and the unlabeled fraction; and

c) separating the labeled fraction from the unlabeled fraction forming a separated labeled fraction and a separated unlabeled fraction, wherein at least about 40% of the labeled fraction contains viable sperm cells that can be used for in vivo or in vitro fertilization.