# Patent

## Title
Gender-specific separation of sperm cells and embryos

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2008/086833, filed Dec. 15, 2008, which claims the benefit of priority to U.S. Provisional Patent Application No. 60/013,901, filed Dec. 14, 2007, the entire disclosure of which is herein incorporated by reference for any purpose.


INTRODUCTION
The production of offspring of a predetermined sex, or in a predetermined sex ratio, is desirable in a number of industries, including animal husbandry. The gender-specific separation of sperm cells or embryos may facilitate the production of offspring having a predetermined sex. Separated sperm cells may be used in artificial insemination or in vitro fertilization to produce zygotes that develop into organisms of a predetermined sex. However, techniques to produce populations of sperm cells or embryos that are sufficiently gender enriched are lacking.
SUMMARY
In one aspect, the invention provides a method for separating a population of sperm cells by contacting the population with a labeled oligonucleotide moiety capable of binding a gender-specific tandem repeat sequence that occurs in a portion of the population. The labeled sperm cells are then separated from the unlabeled sperm cells.
In another aspect, the invention provides a sperm cell or embryo having a gender-specific tandem repeat sequence and a labeled oligonucleotide moiety, such as a triplex forming oligonucleotide or a peptide nucleic acid, bound to the gender-specific sequence.
In another aspect, the invention provides a population of sperm cells or embryos having a gender-specific tandem repeat sequence occurring on the X or the Y chromosome, and a portion of the cells having a labeled oligonucleotide moiety, such as a triplex forming oligonucleotide or a peptide nucleic acid, bound to the gender-specific sequence.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation showing the location of gender-specific tandem repeat sequences (GSTRSs) and target sequences on a chromosome.
FIG. 2 includes histograms of boar sperm cells with CY3-labeled TFO (Sequence C) targeting the porcine Y chromosome GSTRS of SEQ ID NO: 1. FIG. 2A depicts data from sorted samples with CY3-labeled TFO and FIG. 2B depicts data from the negative control.



DETAILED DESCRIPTION
The invention relates to the generation of sperm cell fractions or embryos that are enriched for the X or the Y chromosome. In one embodiment, the invention provides methods for separating sperm cells that contain a labeled oligonucleotide moiety bound to a gender-specific tandem repeat sequence or a complement of a gender-specific tandem repeat sequence. The oligonucleotide moieties suitably bind in sufficient numbers to a region of the chromosome to generate a detectable signal that can be used as a basis for distinguishing and separating cells that contain the gender-specific tandem repeat sequence from those that do not. The invention further provides a method for the separation of sperm cells carrying an X chromosome from sperm cells carrying a Y chromosome. The gender-enriched sperm cell fractions can be used to fertilize ova to produce offspring of a predetermined sex. The invention further provides a method for selection of embryos carrying an X chromosome or embryos carrying a Y chromosome.
As used herein, a “gender-specific tandem repeat sequence,” or “GSTRS” is a non-autosomal chromosome sequence that is repeated on either the Y chromosome or the X chromosome, but not both. Multiple GSTRSs occur in a region of the X or Y chromosome, as shown schematically in FIG. 1. The GSTRS may occur anywhere on the X or Y chromosome. In some embodiments, the GSTRS targets of the invention occur at or near the termini of the chromosome. The gender-specific tandem repeat sequence may comprise at least about 50 nucleotides, at least about 100 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 3,000 nucleotides, or at least about 4,000 nucleotides, and less than about 10,000 nucleotides, less than about 9,000 nucleotides, less than about 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, or less than about 5,000 nucleotides. Suitably there are less than about 50,000 nucleotides, about 10,000 nucleotides, about 5,000 nucleotides, about 3,000 nucleotides, about 2,000 nucleotides, about 1,000 nucleotides, about 500 nucleotides, about 300 nucleotides, about 100 nucleotides, about 10 nucleotides, about 1 nucleotide, or zero nucleotides between each unit of the repeated GSTRS. The GSTRS does not have to be repeated as exactly the same sequence, and some variation in the repeated sequences is possible without effecting the scope of the invention. The units of repeated GSTRSs may share at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with each other. Percent identity may be determined using algorithms used in BLASTn or MEGABLAST programs, which may be used to obtain sequences homologous to a reference polynucleotide, as is known in the art. Algorithms used for sequence alignment are described by Tatiana A. Tatusova, Thomas L. Madden (1999), FEMS Microbiol Lett. 174:247-250. The GSTRS may be repeated at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 750 times, or at least about 1000 times on a chromosome.
For each GSTRS, an oligonucleotide moiety may be selected to bind to the GSTRS or a complement of the GSTRS. As depicted schematically in FIG. 1, the oligonucleotide moiety may target a shorter target sequence within the GSTRS. As used herein, “target sequence” is a segment of DNA within the GSTRS, wherein the oligonucleotide moiety binds the target sequence or the complement of the target sequence. The target sequence may include at least about 4, at least about 6, at least about 8, at least about 10, at least about 12, or at least about 14 nucleotides. The target sequence may include less than about 100, less than about 90, less than about 80, less than about 70, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 16 nucleotides. The oligonucleotide moiety may bind to at least about 4 nucleotides, at least about 5 nucleotides, at least about 6 nucleotides, at least about 9 nucleotides, at least about 12 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, or at least about 35 nucleotides of the GSTRS or the complement of the GSTRS. The oligonucleotide moiety may bind to less than about 100 nucleotides, less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, or less than about 20 nucleotides of the GSTRS or the complement of the GSTRS.
Suitable GSTRSs may be selected by searching public databases for DNA sequences that are highly repetitive on only the X or the Y chromosome. Suitable target sequences within the GSTRS may be selected by scanning the GSTRS for consecutive purines or consecutive pyrimidines, for example, homopurine or homopyrimidine sequences. Homopurine or homopyrimidine sequences facilitate binding of oligonucleotide moieties such as triplex-forming oligonucleotides (TFOs) to the major groove of duplex DNA to form a triplex. The target sequence within the gender-specific tandem repeat sequence may include, but is not limited to, homopurine or homopyrimidine sequences, as in certain embodiments, oligonucleotide moieties are capable of binding DNA of any sequence.
In some embodiments, the target sequence is itself a repeated unit within the GSTRS. The GSTRS may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 7, at least about 10, at least about 15, at least about 50, at least about 100, or at least about 200 repeated units of target sequence. A GSTRS having a higher number of repeated units will facilitate binding of more oligonucleotide moieties to the GSTRSs. Suitably, at least about 5, at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 5,000, at least about 25,000, or at least about 50,000 oligonucleotide moieties bind to the GSTRSs.
In some embodiments, more than one target sequence may be selected within the GSTRS, or a complement of the GSTRS. A GSTRS having a higher number of target sequences will facilitate binding of more oligonucleotide moieties to the GSTRSs. In other embodiments, more than one type of oligonucleotide moiety may be selected to bind the GSTRS or a complement of the GSTRS.
The oligonucleotide moiety that binds to the target sequence is a peptide nucleic acid (PNA) or a triplex forming oligonucleotide (TFO).
A PNA is two or more purine or pyrimidine bases linked to a peptide backbone. The PNA backbone is made of repeating N-(1-aminoethyl)-glycine units linked by peptide bonds, with the bases linked to the backbone by methylene carbonyl bonds. The neutral backbone of PNAs facilitates binding DNA with reduced charge repulsion relative to the phosphodiester backbone. PNAs bind duplex DNA by forming Watson-Crick pairs with the target sequence or complement of the target sequence, and creating a single-stranded DNA loop. PNAs and methods for generating PNAs are described in U.S. Pat. No. 5,539,082 and Pellestor et al. (Human Reproduction (2004) volume 19, pages 1946-1951), which are incorporated herein by reference in their entireties. In some embodiments, the PNA includes a detectable label attached to the sequence by an ester or other linkage, as further described below.
A TFO is a sequence of at least two nucleotides capable of binding within the major groove of a DNA double helix in a sequence-specific manner. A TFO binds to DNA bases and/or RNA bases to form a triple helical structure. For example, a TFO may target a homopurine or homopyrimidine sequence in the DNA double helix to form a triple helix. Purine TFOs and pyrimidine TFOs each bind purine sequences. Purine TFOs bind in the major groove of duplex DNA using reverse Hoogsteen hydrogen bonds and run antiparallel to the purine sequence. Pyrimidine TFOs bind in the major groove of duplex DNA using Hoogsteen hydrogen bonds and run parallel to the purine sequence (see Li, J. et al. (2003) Journal of the American Chemical Society volume 125, pages 2084-2093, which is incorporated herein by reference in its entirety). Suitable TFOs and methods of generating TFOs are described in U.S. Pat. Nos. 5,176,996 and 5,962,426, which are incorporated herein by reference in their entireties. TFOs may be synthesized by standard phosphoramidite synthesis or other techniques known in the art.
OligoTRIPs are a type of synthetic TFO that recognize and bind the major groove of a DNA duplex of any sequence and do not require homopurine or homopyrimidine target sequences. OligoTRIPs can target a sequence comprising both purines and pyrimidines because the glycosidic bond of the OligoTRIP backbone is near the center of the major groove and perpendicular to the Watson-Crick hydrogen bonds of the target duplex DNA. The location of the glycosidic bond and the capacity of OligoTRIP bases to recognize A:T versus T:A and C:G versus G:C allow the OligoTRIP bases to bind purines on either side of the major groove of the duplex DNA. OligoTRIPs may be synthesized from one or more heterocyclic OligoTRIP monomers, such as AntiAT-F, AntiTA, AntiGC, or AntiCG. Suitable OligoTRIPs and methods of generating OligoTRIPs are described in U.S. Pat. No. 5,844,110 and U.S. Patent Application Publication No. US 2006/0281907, which are incorporated herein by reference in their entireties. OligoTRIPs incorporate a quinazoline or quinoline base moiety, such as 2-amino-6-fluoro-quinoline or 2-amino-quinazoline. In contrast, non-OligoTRIP TFOs, herein referred to as “naturally occurring TFOs,” have purine or pyrimidine base moieties (for example A, C, T or G) such as are found in naturally occurring polynucleotides. The sugar or base moieties of naturally occurring TFOs may be modified, for example by methylation, to enhance their capacity to bind target sequences. Suitably, an internal cytosine may be methylated as 5-methyl cytosine.
The oligonucleotide moiety that binds to the GSTRS may include a label that is detectable when bound to the gender-specific tandem repeat sequence. Suitable labels include, but are not limited to, dyes, fluorescent molecules such as CY3 or CY5, molecules of heavy density such as gold or iron, magnetic molecules, nanoparticles, picoparticles, or any combination thereof. The labeled oligonucleotide moiety binds in sufficient numbers to the GSTRSs to produce a detectable signal. The signal may be detectable by any suitable method including, but not limited to, centrifugation, fluorescence, luminescence, microscopy, magnetic force, densitometry, or combinations thereof. Methods of coupling labels to oligonucleotides are known in the art and can be adapted for coupling to the oligonucleotide moieties described herein.
In other embodiments, oligonucleotide moieties may be labeled with labels that are active for fluorescence resonance energy transfer (FRET). Some oligonucleotide moieties may be labeled with a FRET donor, and others may be labeled with a FRET acceptor. Excitation of the donor label may excite the acceptor label, and cause the acceptor label to fluoresce. FRET may thus be used to enhance or differentiate the signal of the labeled oligonucleotide moieties bound to GSTRSs in proximity on the chromosome and improve signal to noise ratio. For example, two oligonucleotide moieties can be designed to bind to a target sequence so that the oligonucleotide moieties are located close to each other after binding to the target sequence, e.g., a first oligonucleotide moiety may be designed to bind base pairs 1 to 12 and a second oligonucleotide moiety may be designed to bind base pairs 13 to 24 of a target sequence 24 base pairs in length. When the two different oligonucleotide moieties are labeled with suitable dye molecules, for example a cyan fluorescent protein (CFP) as donor and yellow fluorescent protein (YFP) as acceptor, FRET may be used. The labeled cells may be excited with light of a suitable wavelength. For example, if excited with a wavelength of 440 nm, CFP will emit light at 480 nm wavelength which overlaps with the excitation wavelength of YFP, and will lead to a YPF signal emission peak at 535 nm when both oligonucleotide moieties are close together.
In another embodiment, the label may suitably be a molecule or atom attached to the oligonucleotide moiety that enhances activation or deactivates physiological process of the cell, and may be toxic and/or facilitates destruction, incapacitation or inactivation of the cell when bound to a GSTRS. For example, a cell toxin when attached to the GSTRS, may cause the cell to die, may facilitate impairment of the functioning of the cell, may disrupt the cell physiologically, or may impair cellular integrity, so that the cell becomes unviable or incapacitated. Mechanisms through which the label may affect the cell include, without limitation, increase intra-cellular pH, accumulation of cell toxins, induction of selective phototoxicity, cell death through the action of electromagnetic waves on the label and combinations thereof. The Enriched sperm cell fractions may be thus be generated without needing to separate a viable population of labeled cells from a viable population of unlabeled cells. Such a label may be used in conjunction with, or independently from, one or more detectable labels bound to the same or other oligonucleotide moieties.
Suitably, the molecule or atom that facilitates destruction or incapacitation of the cell functions effectively when in proximity to other labels, which labels may be the same or different, and which may each be attached to separate oligonucleotide moieties, as would occur upon binding of the oligonucleotide moiety to the GSTRS.
Labels may also be used which regulate the capacitation of sperm cells containing a GSTRS. Accordingly, the timing at which a labeled sperm cell containing the GSTRS has the capacity to fertilize an egg may be controlled. For example, a sperm cell may be incapacitated in its ability to fertilize an oocyte, by inducing premature capacitation. Fertilization of an egg can then be delayed by an appropriate amount of time, such that the labeled fraction of cells in the population are unable to fertilize the egg.
Suitable labels which may be used include, for example, noble metals such as silver, gold, platinum, palladium, rhodium, and iridium, and alloys and molecules thereof, as well as magnetic compounds. Suitably these labels may be attached as pico particles or nanoparticles. Cells labeled with such metals or compounds may subsequently be exposed to electromagnetic radiation, such as radiowaves, which may heat and/or excite the label resulting in the viability of the cell being impaired or reduced. Other suitable labels include calcium or calcium-containing compounds, calcium/ion pump activators, hydrogen ion/pH pump activators, organic compounds with alcohol groups, acids, and denaturing enzymes such as trypsin.
Labels may be attached to oligonucleotides using techniques known in the art for generally coupling molecules to oligonucleotides.
In a further embodiment, methods for distinguishing and separating sperm cells or embryos that contain an oligonucleotide moiety bound to a GSTRS. In some embodiments, the sperm cells or embryos are mammalian. Suitably, the sperm cells or embryos are mammalian, piscian or avian, or from vertebrates. The sperm cells may be of porcine, equine, bovine, ovine, caprine, feline, canine, or human origin. In other embodiments, the sperm cells or embryos are piscian or avian. As used herein, a “population” of sperm cells or embryos means at least two sperm cells or two embryos.
In a first step of the method to generate gender-enriched sperm cell fractions, sperm cells are contacted with the labeled oligonucleotide moiety. In some embodiments, the sperm cell is permeabilized to facilitate entry of the oligonucleotide into the sperm cells and access to the GSTRS. The sperm cells may be permeabilized by any suitable technique, including but not limited to, osmotic pressure, electroporation, liposomes, permeating peptides, a modified (for example increased or decreased) temperature or combinations thereof. In other embodiments, the labeled oligonucleotide moiety is passively or actively transported into the cell. The oligonucleotide moiety may further include a transport moiety, such as a transport peptide, that facilitates or mediates active uptake of the oligonucleotide moiety into the sperm cell. Suitable transport peptides are commercially available from AnaSpec (San Jose, Calif., U.S.A.) and include Arg9, TAT, and Cys-TAT. Transport peptides compatible with the ergothionine transporter may also be used.
Once the oligonucleotides are bound to the repeated DNA sequence, the sperm cells may be separated. The clustering of the labeled oligonucleotide moiety in the region of the GSTRS produces a signal that is detectable and enables cells that contain the GSTRS to be distinguished from cells that do not contain the GSTRS. Once labeled, the sperm cells may be detected and separated. Suitable methods for separating sperm cells include, but are not limited to, centrifugation, magnetic force, flow cytometry, densitometry, or any combination thereof. Suitably, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells in the separated population of sperm cells comprise a labeled oligonucleotide moiety bound to the GSTRS. The population of sperm cells may be separated into a labeled fraction that contains the GSTRS, and an unlabeled fraction that does not contain the GSTRS.
In one embodiment, the labeled fraction includes sperm cells containing an X chromosome labeled with the oligonucleotide, and the unlabeled fraction includes sperm cells containing Y chromosome not labeled with oligonucleotide. In another embodiment, the labeled fraction includes sperm cells containing a Y chromosome labeled with the oligonucleotide, and the unlabeled fraction includes sperm cells containing an X chromosome not labeled with oligonucleotide. Suitably, a fraction may contain sperm cells wherein at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the sperm cells comprise an X chromosome. Alternatively, a fraction may contain sperm cells wherein at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the sperm cells comprise a Y chromosome.
The separated fractions suitably contain viable sperm cells. As used herein, “viable” refers to a sperm cell that is able to fertilize an egg to produce an embryo. Suitably, a separated sperm fraction contains sperm wherein at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of the sperm cells are viable.
A gender-enriched sperm cell fraction may be used to fertilize an egg in vitro or in vivo. Fertilization of an egg may be accomplished, for example, via artificial insemination, including, but not limited to, intra-vaginal, intra-cervical, intra-uterine or surgical insemination, or by intracytoplasmic sperm injection (ICSI). A labeled fraction or unlabeled fraction may be used for in vivo or in vitro fertilization. The fertilized egg may be allowed to develop to produce an embryo of predetermined sex.
In a further embodiment, the invention provides methods for determining the sex of embryos. Labeled oligonucleotide moieties designed to bind to a GSTRS as described above may be incubated with embryos, enter the embryos, and bind the GSTRS. As with sperm cells, the embryos may be permeabilized to facilitate entry of the labeled oligonucleotide moiety into the embryos. One or more cells from the embryo may also be removed or biopsied and permeabilized to facilitate entry of the labeled oligonucleotide. The sex of the biopsied cells may then be correlated with the embryo from which the cells were removed. Once the oligonucleotide moiety is bound to the GSTRS, the embryos may be viewed under a dissecting microscope or fluorescent microscope to distinguish embryos that contain the GSTRS from those that do not contain the GSTRS. As with sperm cells as described above, the population of embryos may be separated into a labeled fraction that contains the GSTRS, and an unlabeled fraction that does not contain the GSTRS.
The following examples are provided to assist in a further understanding of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope of the appended claims.
EXAMPLES
Example 1: Porcine GSTRSs, Target Sequences, and Corresponding Oligonucleotide Moieties
Two PNAs designated “Sequence A” and “Sequence B” each bind the target sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is a 16-nucleotide sequence at nucleotide positions 344 to 359 of the GSTRS depicted in SEQ ID NO: 1. SEQ ID NO: 1 occurs on the porcine Y chromosome and has sequence Accession number X12696 (McGraw et al. (1988) Nucleic Acids Research, volume 16, page 10389). PNAs may be designed to bind to a specific sequence using materials and information commercially available from Applied Biosystems. Sequence A and Sequence B were each custom synthesized with a CY3 fluorescent molecule attached to the 5′-end by an ester bond. Sequences A and B were custom-ordered from Bio-Synthesis, Inc. (Lewisville, Tex., U.S.A.). PNAs were received as a lyophilized powder, and they were resuspended in ultrapure water and stored in aliquots at −20° C. and at −80° C.
A TFO designated “Sequence C”, which binds the target sequence shown in SEQ ID NO: 2, was custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond. A TFO designated “Sequence D” was designed to bind to the target sequence shown in SEQ ID NO: 3 and was custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond. SEQ ID NO: 3 is a 20-nucleotide sequence at nucleotide positions 3681 to 3700 of the GSTRS shown in SEQ ID NO: 1.
A 205 base pair GSTRS was identified on the porcine X chromosome at locus AY574041 (SEQ ID NO: 4). Two TFOs designated “Sequence H” and “Sequence I” were designed to bind to the target sequence shown in SEQ ID NO: 9. SEQ ID NO: 9 is a 15-nucleotide sequence at the 3′-end of the GSTRS shown in SEQ ID NO: 4. Sequence H was custom synthesized with a FAM fluorescent molecule linked to the 3′-end, and Sequence I was custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond. A PNA designated “Sequence J” was designed to bind to the target sequence shown in SEQ ID NO: 9, and includes a “J base” moiety: 2′-Deoxypseudoisocytidine CEP (ψ-iso-dC). Sequence J was custom synthesized with a BoDIPY FLX (FITC-like) fluorescent molecule linked to the 5′-end by a linker.
A 1399 base pair GSTRS was identified on the bovine X chromosome at locus V00125 (SEQ ID NO: 5) with sequence Accession number V00125. A TFO designated “Sequence E” was designed to bind to the target sequence shown in SEQ ID NO: 6. SEQ ID NO: 6 is a 16-nucleotide sequence at nucleotide positions 1157 to 1172 of the GSTRS shown in SEQ ID NO: 5. Sequence E was custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond.
A somatic tandem repeated DNA sequences was identified on porcine chromosome 1 with sequence Accession number X51555 (SEQ ID NO: 7). It is a 313 base pair DNA sequence that is repeated approximately 3000 to 6000 times. Two TFOs designated “Sequence F” and “Sequence G” were each designed to bind to the target sequence shown in SEQ ID NO: 8. SEQ ID NO: 8 is a 14-nucleotide sequence at nucleotide positions 120 to 133 of the tandem repeat DNA sequence shown in SEQ ID NO: 7. Both Sequence F and Sequence G were each custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond. This somatic DNA sequence (SEQ ID NO: 7) was used as a negative control for experiments.
Examples of DNA sequences of porcine GSTRSs, target sequences, and corresponding oligonucleotides moieties are shown in Table 1 below.









TABLE 1





Name
Description
DNA Sequence, 5′ → 3′







SEQ ID NO: 1
GSTRS on porcine Y
See SEQ ID NO: 1.



chromosome.



 


SEQ ID NO: 2
Target sequence near the 5′-
5′-GAGAGAGAGA GAGAGA-3′



end of the GSTRS shown in




SEQ ID NO: 1.



 


SEQ ID NO: 3
Target sequence near the 3′-
5′-TCTAGAGAAG GAGGAGGATT-



end of the GSTRS shown in
3′



SEQ ID NO: 1.



 


SEQ ID NO: 4
GSTRS on porcine X
See SEQ ID NO: 4.



chromosome.



 


SEQ ID NO: 5
GSTRS on bovine X
See SEQ ID NO: 5.



chromosome.



 


SEQ ID NO: 6
Target sequence in the
5′-AGGAGGGGAG AAAGGG-3′



GSTRS shown in




SEQ ID NO: 5.



 


SEQ ID NO: 7
Tandem repeat sequence on
See SEQ ID NO: 7.



porcine chromosome 1.



 


SEQ ID NO: 8
Target sequence in the
5′-TCCGCCTCCT CCCT-3′



tandem repeat sequence




shown in




SEQ ID NO: 7.



 


SEQ ID NO: 9
Target sequence at the 5'-end
5′-CTCCCTTCTG TCTTT-3′



of the GSTRS shown in




SEQ ID NO: 4.



 


Sequence A
PNA; binds to target
5′-TCTCTCTCTC TCTCTC-3′


SEQ ID NO: 12
sequence shown in




SEQ ID NO: 2.



 


Sequence B
PNA; binds to target
5′-TCTCTCTCTC TC-3′


SEQ ID NO: 13
sequence shown in




SEQ ID NO: 2.



 


Sequence C
TFO; binds to target
5′-CTCTCTCTCT CTCTCT-3′


SEQ ID NO: 14
sequence shown in




SEQ ID NO: 2.



 


Sequence D
TFO; binds to target
5′-TCTCTTCCTCCTCCT-3′


SEQ ID NO: 15
sequence shown in




SEQ ID NO: 3.



 


Sequence E
TFO; binds to target
5′-TCCTCCCCTC TTTC-3′


SEQ ID NO: 16
sequence shown in




SED ID NO: 6.



 


Sequence F
TFO; binds to target
5′-TCCCTCCTCC TCCT-3′


SEQ ID NO: 17
sequence shown in




SEQ ID NO: 8.



 


Sequence G
TFO; binds to target
5′-GGTGGTGGGT-3′


SEQ ID NO: 18
sequence shown in




SEQ ID NO: 8.



 


Sequence H
TFO; binds to target
5′-GTGGGTTGT-3′



sequence shown in




SEQ ID NO: 9.



 


Sequence I
TFO; binds to target
5′-GTGGGTTGT-3′



sequence shown in




SEQ ID NO: 9.



 


Sequence J
PNA; binds to target
5′-CTCCCTTCTG TCTTTAT-3′


SEQ ID NO: 19
sequence shown in




SEQ ID NO: 9.



 


Sequence K
PNA; binds to target
5′-AGC CCT GTG CCC TG-3′


SEQ ID NO: 20
sequence shown in




SEQ ID NO: 11.









Example 2: Generation of a CY3-Labeled Triplex Forming Oligonucleotide (TFO) Conjugate
Triplex forming oligonucleotides (TFOs) were obtained from Unimed Medical Center (University of Nebraska-Omaha, Omaha, Nebr., U.S.A.) or Integrated DNA Technologies (Coralville, Iowa, U.S.A.). A labeled CY3-TFO conjugate was made by linking a CY3 dye, commercially available from Applied Biosystems (Foster City, Calif., U.S.A.), to the 5′-end of the TFO prepared as described in Example 1 (designated Sequence C) with an ester linkage. The internal cytosines were methylated.
Example 3: Method of Labeling Fixed Boar Sperms Cells with CY3-PNA Conjugate
Freshly-ejaculated boar semen or thawed boar semen (about 100 million sperm cells) was added to 10 mL of phosphate-buffered saline (PBS). The suspension was centrifuged for 5 minutes at 800×g. The pellet was resuspended in 1 mL of 3 M NaOH. The suspension was incubated at room temperature for 5 minutes and centrifuged for 5 minutes at 800×g. The pellet was resuspended in 2 mL of PBS and centrifuged for 5 minutes at 800×g. The pellet was resuspended in PBS or phosphate buffer (PB) to obtain a final concentration of 10 million sperm cells per mL of PBS.
After pre-treatment of the sperm cells, CY3 labeled-PNA as prepared in Example 1 (designated Sequence A) was incubated with the sperm cells at a final PNA concentration of 50 ng/mL for 7 minutes at 75° C. The suspension was allowed to cool to room temperature and then incubated at room temperature for 45 minutes. Sperm cells were centrifuged for 5 minutes at 800×g, the pellet was resuspended in PBST (PBS with 0.05% Tween 20), and the suspension was incubated for 20 minutes at 45° C. The sperm cells were centrifuged for 5 minutes at 800×g, and the pellet was resuspended in PBS or PB. CY3 labeled (Sequence A)-PNA-treated sperm cells (4 μL) were viewed under a Zeiss AxioSkop fluorescence microscope. DAPI stain was optionally added to the sample just before observation with the microscope. Selective binding of the CY3 labeled-PNA to the Y chromosome of fixed boar semen was observed. Fixed boar sperm cells pretreated with NaOH and RNase A and incubated with Y-chromosome specific CY3-PNA Y90-1 stained Y chromosomes red. Somatic porcine chromosomes treated with Y-chromosome specific CY3-PNA Y90-1 were stained red. Somatic porcine chromosomes stained with DAPI that binds DNA and RNA and were stained blue. A merged image of somatic porcine chromosomes stained with DAPI and CY3-PNA Y90-1 was generated. Y-chromosomes appeared to be stained pink, indicating selective binding of CY3-PNA Y90-1 to the Y-chromosomes.
We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally-located, round fluorescent label in the sperm head.
As a control, freshly-ejaculated boar semen was prepared and permeabilized as described above. A CY3-PNA conjugate with base sequence (CCCTAA)3, commercially available from Applied Biosystems (Foster City, Calif., U.S.A.) that binds to the telomeres of all mammalian chromosomes was incubated with the resuspended sperm cells in PBS at a final PNA concentration of 1 ng/μL for 1 to 18 hours at room temperature. CY3-PNA (CCCTAA)3-treated sperm cells (4 μL) were viewed under a Zeiss AxioSkop fluorescence microscope. Selective binding of CY3-PNA (CCCTAA)3 to all porcine chromosome telomeres of fixed boar semen was observed. Chromosomes stained with 4′,6-diamidino-2-phenylindole (DAPI) that non-specifically binds DNA and RNA appeared blue. In contrast, CY3-PNA (CCCTAA)3 stained chromosomes pink.
Example 4: Method of Labeling Fixed Boar Sperm Cells with CY3-TFO Conjugate
Freshly-ejaculated boar semen or thawed boar semen (about 100 million sperm cells) was added to 10 mL of PBS. The suspension was centrifuged for 5 minutes at 800×g, and the pellet was resuspended in 1 mL of 3 M NaOH. The suspension was incubated at room temperature for 5 minutes and centrifuged for 5 minutes at 800×g. The pellet was washed in 2 mL of PBS and centrifuged for 5 minutes at 800×g. The pellet was resuspended in PBS or PB to obtain a final concentration of 10 million sperm cells per mL of PBS.
After pre-treatment of the sperm cells, 2 mM MgCl2 was added to the sperm cell suspension. The suspension was incubated with the fluorescently labeled TFO conjugate at a final concentration of 50 ng/mL at 60 to 70° C. for 20 minutes to 1 hour. The suspension was centrifuged at room temperature at 800×g for 5 minutes, the supernatant was aspirated, and the pellet was resuspended in PBS or PB. CY3-TFO-treated sperm cells were viewed under a Zeiss AxioSkop fluorescence microscope to confirm that the chromosomes were labeled with CY3-TFO conjugate. Flow cytometry was used to analyze the semen. Histograms of boar sperm cells with CY3-labeled TFO (of Sequence C) targeting the porcine Y chromosome GSTRS of SEQ ID NO: 1 are shown in FIGS. 2A and 2B. FIG. 2A depicts flow data from sorted samples with CY3-labeled TFO and FIG. 2B depicts flow data from the negative control.
Example 5: Separation of Fluorescently Labeled Boar Sperm Cells Via Flow Cytometry
Boar sperm cells were permeabilized by adding approximately 100 million sperm cells to 10 mL of PBS. The suspension was centrifuged for 5 minutes at 800×g, and the pellet was resuspended in 1 mL of 1 to 3 M NaOH. The suspension was incubated at room temperature for 5 minutes and centrifuged for 5 minutes at 800×g. The pellet was washed in 2 mL of PBS and centrifuged for 5 minutes at 800×g. The pellet was resuspended in PBS or PB to obtain a final concentration of 10 million sperm cells per mL of PBS. After pre-treatment of the sperm cells, 2 mM MgCl2 was added to the sperm cell suspension. The suspension was incubated at 60 to 70° C. for 20 minutes to 1 hour.
A PNA as prepared in Example 1 (Sequence A; labeled with CY3) was incubated with the sperm cells at 1 ng/μL PNA for 1 to 18 hours at room temperature. Boar sperm cells were then separated using a FACSVantage SE with DiVa option flow cytometer (BD Biosciences, San Jose, Calif., U.S.A.) with 100 mW of 488 nm light from a Coherent INNOVO 90C Argon ion laser. A 100 μm nozzle tip was used at a sheath pressure of 12 psi. The sheath fluid used was sterile Dulbecco's Phosphate Buffered Saline (DPBS, without Ca2+ or Mg2+, Sigma-Aldrich, St. Louis, Mo., U.S.A.). Detectors used included FSC-A for forward scatter, SSC-A for side scatter, FL1-A with a 530/30 nm bandpass filter to detect any auto-fluorescent material, FSC-W for doublet-discrimination, and FL2-A CY3 detector with a 585/42 nm bandpass filter to detect the PNA with CY3 fluorescent label. A flow cytometry histogram illustrating the separation of labeled and unlabeled boar sperm cells demonstrated selective binding of CY3-PNA (Sequence A) to the Y chromosome and separation of sperm with X chromosome from sperm with Y chromosome. At least 85% of the cells in the labeled fraction are expected to contain the Y chromosome. At least 85% of the cells of the unlabeled fraction are expected to contain the X chromosome.
Example 6: Separation of Fluorescently Labeled Viable Sperm Cells Via Flow Cytometry
Semen will be resuspended in semen extender (AndroHep CellGuard for boar sperm, commercially available from Minitube of America, Verona, Wis., U.S.A.) to give approximately 1×107 cells per mL. 1 ng of CY3-PNA conjugate of Example 1 (Sequence A) will be added to 0.6 mL of sperm cell suspension. The suspension will be placed into a 0.4 cM electrode Bio-Rad cuvette (Bio-Rad, Hercules, Calif., U.S.A.), and the cells will be permeated using a pulsive Gene Pulser (Bio-Rad) set to 400 volts at 25 uFD capacitance after 5 seconds. The electroporation pulse will be repeated. The suspension will be incubated at 18° C. for 1 to 24 hours. Uptake of PNA into the sperm will be verified by fluorescence microscopy.
The labeled sperm cells will be separated from the unlabeled sperm cells under flow using a FACSVantage SE with DiVa option flow cytometer with conditions as in Example 5. Selective binding of the PNA to the Y chromosome in the cells of the labeled fraction will be demonstrated by a flow cytometry histogram. At least 70% of the cells in the labeled fraction are expected to contain the Y chromosome. At least 70% of the cells of the unlabeled fraction are expected to contain the X chromosome.
Example 7: In Vitro Fertilization of Porcine Eggs with X or Y Chromosome-Enriched Boar Semen
Viable boar sperm cell fractions labeled with CY3-PNA or unlabeled, as described in Example 6, were used to fertilize porcine eggs. About 1.5 to 2 hours before preparing the semen, one plate or dish containing 5 to 10 mL of TALP media and one plate or dish containing 5 to 10 mL of FERT media (TALP+caffeine) were prepared and placed in an incubator 38.5° C. for at least 1.5 hours to equilibrate. Additionally, approximately 30 mL of semen saline (0.9% saline+BSA) was placed in a hood to warm to room temperature. Sperm vision counting chambers were warmed.
To prepare the semen, 2 to 3 mL of the X or Y chromosome-enriched sperm cell fraction was brought up to 10 mL with semen saline (0.9% saline+BSA). The suspension was centrifuged at 800×g for 3 minutes. The semen saline was pulled down to the sperm pellet, the volume brought up to 10 mL with fresh semen saline, the pellet resuspended in fresh saline, and the suspension centrifuged. The washing procedure may be repeated for a total of three times. The final sperm pellet was resuspended in 3 mL of TALP, mixed gently, and a small sample was removed for subsequent sperm motility and concentration determination.
To prepare frozen-thawed X or Y chromosome-enriched sperm cell fraction, a frozen straw of semen (0.5 cc) was placed in a 50° C. water bath for 10 seconds. The thawed sperm was then layered over a density gradient and centrifuged at 350×g for 10 minutes. The pellet was washed once in 2 mL of Cellguard (Minitube, Verona, Wis., U.S.A.) and centrifuged at 200×g for 10 minutes. The pellet was diluted and mixed gently in 1 mL of TALP media, and a small sample was removed for subsequent sperm motility determination. Sperm motility and concentration was determined using Sperm Vision (Minitube of America, Verona, Wis., U.S.A).
To fertilize oocytes, 10 μL of sperm in FERT media (at a concentration of 2.5×105 sperm/mL) was added to a 500 μL well containing 50 oocytes. In vitro fertilization of porcine oocytes is also described in Rath et al. (J. Anim. Sci. 77:3346-3352 and Long, et al. (1999) Theriogenology 51:1375-1390), each of which is incorporated herein by reference in its entirety.
Example 8: Generation of a CY3-Labeled Peptide Nucleic Acid (PNA) Conjugate and Use to Identify Male and Female Sperm
Synthetic DNA mimics conjugated to a fluorescent dye were used for in situ detection of Y chromosomes in metaphase preparations of bovine somatic cells and spermatozoa. Using male bovine somatic cells and the Y-chromosome as a template, a synthesis a CY3-conjugated PNA was designed and custom synthesized.
A PNA designated “Sequence K” was designed to bind to the target sequence shown in SEQ ID NO: 11. SEQ ID NO: 11 is a 14-nucleotide sequence at nucleotide positions 562 to 576 of the GSTRS shown in SEQ ID NO: 10. SEQ ID NO. 10 is a bovine Y chromosome sequence thought to be repeated 60,000 times (Perret, J. et al., 1990. A polymorphic satellite sequence maps to the pericentric region of the bovine Y chromosome; Genomics Vol 6 (3) pp 482-490). The PNA probe designated “Sequence K” was custom synthesized with a CY3 fluorescent molecule linked to the 5′-end by an ester bond: CY3-OO-AGC CCT GTG CCC TG
Flow cytometry generated sexed bull sperm were evaluated with the PNA probe (Sequence K) for accuracy of scoring. By testing different labeling conditions, it was found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on the Y-chromosome. The Y sorted sperm population showed labeling with the PNA probe in 104 signals on sperm heads out of 118 counted. The X sorted population showed labeling with the PNA probe in 8 signals on sperm heads out of 119 counted. In other tests, no signals were present when chromosomes of bovine female somatic cells were incubated with the PNA probe.
The PNA signals present in about 50% of sperm were found to consist of a single, centrally-located, round fluorescent dot in the sperm head. Unsorted bull sperm provided 23 signals out of 43 sperm heads (53.4%). The PNA probe was also found to produce signal in male bovine somatic cell lines and in embryos.
Example 9: Sex Determination of Bovine Embryos Using Bovine Blastomeres Recovered Via Biopsy
A biopsy (n=5 to 8 blastomeres, pellucida-free) was taken from a blastocyst-stage embryo washed with phosphate buffered saline (PBS) and transferred onto a plastic slide. Cells were completely air-dried and heated on the slide to 60° C. for 1 minute. Cells were fixed by immersion in a solution containing 75% methanol and 25% acetic acid for 5 minutes. 2 μL of Buffer 1 containing 0.2M KCl and 0.2M NaOH, with the final pH being adjusted to 13.0 with 3 M NaOH, was added for 20 seconds, before 250 μL of a Tris-EDTA buffer with 10 mM KCl was added to neutralize Buffer 1. The slide was drained and to the cells were added 250 μL of the peptide nucleic acid probe SEQUENCE K conjugated to a fluorescent dye (CY-3) at the 5′ end in 50% water/50% DMSO, which targets the unique Y-chromosome specific sequence SEQ ID NO. 11. After 5 minutes of incubation the cells were washed and 60 μL, mounting medium containing SlowFade (commercially available from Invitrogen) and DAPI (a DNA stain). Y-chromosomes were detected as a bright spot within the blastomere nuclei. The absence of signal indicated female embryonic DNA. After approximately 75 minutes of incubation with the probe on the slide the embryo gender was detected. The accuracy of the biopsy sexing procedure was demonstrated by parallel gender determination of the same embryo using an established PCR method designed for the bovine amelogenin locus. Based on 18 in vitro produced bovine embryos generating a result for both assays, there was a 94.4% match ( 17/18) of gender assignment.
It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the foregoing description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## 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.