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US 2014/0182005 A1Published application
Sperm cell separation methods and compositions containing sperm cell targeting ligands for use therein
AssigneeBioCern, Inc.
InventorDavid A. OKSENBERG
Life of this patent
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Abstract
The present invention provides sperm cell targeting ligands, including DNA-binding proteins, that bind target molecules on the surface of, accessible from the surface of, or inside mammalian sperm cells and methods for producing the sperm cell targeting ligands. The sperm cell targeting ligands are used to separate sperm cells based upon sperm cell qualities, such as whether the cells contain X chromosomes or Y chromosomes. The invention also provides methods of sperm cell purification using targeted radiofrequency absorption enhancers and transgenic animals with sex-skewed ejaculate.
Claims118
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with a sperm cell targeting ligand;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said sperm cell targeting ligand.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample with a sperm cell targeting ligand attached to at least one radionuclide;
- (b) introducing into said mammalian sperm sample targeted radio frequency absorption enhancers;
- (c) transmitting a hyperthermia generating radio frequency signal toward the target sperm cells;
- and
- (d) recovering the unbound sperm cells.
2 more elements
A non-human mammal comprising:
- a DNA segment encoding a selfish genetic element (SGE) heterologous to the mammal operably linked to at least one regulatory sequence effective to promote expression of the DNA segment.
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with a dendrimer-oligonucleotide complex;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said dendrimer-oligonucleotide conjugate.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample with a dendrimer-oligonucleotide complex attached to at least one radionuclide;
- (b) introducing into said mammalian sperm sample targeted radio frequency absorption enhancers;
- (c) transmitting a hyperthermia generating radio frequency signal toward the target sperm cells;
- and
- (d) recovering the unbound sperm cells.
2 more elements
A method of separating mammalian sperm cells, comprising the steps of:
- (a) incubating a mixed sperm cell population under conditions which allow for the binding of a labeled dendrimer-oligonucleotide complex to a target molecule found inside said mammalian sperm cells;
- and
- (b) separating said mammalian sperm cells into two or more populations based on the ability of the sperm cells to bind the labeled dendrimer-oligonucleotide complex.
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with a dendrimer-PNA complex;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said dendrimer-oligonucleotide conjugate.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample with a dendrimer-PNA complex attached to at least one radionuclide;
- (b) introducing into said mammalian sperm sample targeted radio frequency absorption enhancers;
- (c) transmitting a hyperthermia generating radio frequency signal toward the target sperm cells;
- and
- (d) recovering the unbound sperm cells.
2 more elements
A method of separating mammalian sperm cells, comprising the steps of:
- (a) incubating a mixed sperm cell population under conditions which allow for the binding of a labeled dendrimer-PNA complex to a target molecule found inside said mammalian sperm cells;
- and
- (b) separating said mammalian sperm cells into two or more populations based on the ability of the sperm cells to bind the labeled dendrimer-PNA complex.
A method for the selective enrichment of a sperm cell population from a suspension of a sperm cell populations containing the target cell population and a non-target cell population, comprising the steps of:
- (a) contacting a magnetizable dendrimer-oligonucleotide complex with a suspension of sperm cell populations, said dendrimer-oligonucleotide complex having affinity for a specific population of sperm cells;
- and
- (b) subjecting the magnetizable dendrimer-oligonucleotide complex to a magnetic field;
- whereby one population of cells binds to said particles so as to be retained, thereby enriching the target cell population.
1 more element
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with a DNA-binding protein;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said DNA-binding protein.
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with at least two DNA-binding proteins;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said DNA-binding proteins.
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting said mammalian sperm cells with a first zinc finger protein conjugated, complexed, or fused with a first fluorescence resonance energy transfer (FRET) enabling marker;
- (b) contacting said mammalian sperm cells with a second zinc finger protein conjugated, complexed, or fused with a second fluorescence resonance energy transfer (FRET) enabling marker;
- (c) subjecting said mammalian sperm cells to conditions which allow for fluorescence resonance energy transfer (FRET) to occur between said first fluorescence resonance energy transfer (FRET) enabling marker and said second fluorescence resonance energy transfer (FRET) enabling marker;
- and
- (d) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said first zinc finger protein and said second zinc finger protein.
2 more elements
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting said mammalian cells with a first zinc finger protein conjugated, complexed, or fused with a first sequence enabled reassembly (SEER) marker;
- (b) contacting said mammalian sperm cells with a second zinc finger protein conjugated, complexed, or fused with a second sequence enabled reassembly (SEER) marker;
- (c) subjecting said mammalian sperm cells to conditions which allow reassembly to occur between said first sequence enabled reassembly (SEER) marker and said second sequence enabled reassembly (SEER) marker;
- (d) detecting reassembly of said first sequence enabled reassembly (SEER) marker and said second sequence enabled reassembly (SEER) marker, and
- (e) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said first zinc finger protein and said second zinc finger protein.
2 more elements
A method of separating mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm cells with a polyamide;
- and
- (b) separating the sperm cells into two or more populations based on the ability of the sperm cells to bind to said polyamide.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample with a DNA-binding protein attached to at least one radionuclide;
- (b) introducing into said mammalian sperm sample targeted radio frequency absorption enhancers;
- (c) transmitting a hyperthermia generating radio frequency signal toward the target sperm cells;
- and
- (d) recovering the unbound sperm cells.
2 more elements
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample with a first zinc finger protein conjugated, complexed, or fused with a first heat enabling nanoparticle;
- (b) contacting said mammalian sperm sample with a second zinc finger protein conjugated, complexed, or fused with a second heat enabling nanoparticle;
- (c) transmitting a hyperthermia generating radio frequency signal toward the target sperm cells to thermally-induce apoptosis triggered by the proximity binding of the zinc fingers;
- and
- (d) recovering the unbound sperm cells.
2 more elements
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample comprising a population of X chromosome-bearing spermatozoa and a population of Y chromosome-bearing spermatozoa with a zinc finger conjugated, complexed, or fused with a nuclease, wherein said zinc fingers are specific for either the population of X chromosome-bearing spermatozoa or Y chromosome-bearing spermatozoa;
- and
- (b) subjecting the bound population to nuclease cleavage at one or more sites, thereby eliminating the bound population from said mammalian sperm sample.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample comprising a population of X chromosome-bearing spermatozoa and a population of Y chromosome-bearing spermatozoa with a plurality of zinc fingers conjugated, complexed, or fused with a nuclease, wherein said zinc fingers are specific for either the population of X chromosome-bearing spermatozoa or Y chromosome-bearing spermatozoa;
- and
- (b) subjecting the bound population to nuclease cleavage at one or more sites, thereby eliminating the bound population from said mammalian sperm sample.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting a mammalian sperm sample comprising a population of X chromosome-bearing spermatozoa and a population of Y chromosome-bearing spermatozoa with a plurality of meganucleases specific for either the population of X chromosome-bearing spermatozoa or Y chromosome-bearing spermatozoa;
- and
- (b) subjecting the bound population to nuclease cleavage at one or more sites, thereby eliminating the bound population from said mammalian sperm sample.
A method of purifying mammalian sperm cells, comprising the steps of:
- (a) contacting the mammalian sperm sample comprising a population of X chromosome-bearing spermatozoa and a population of Y chromosome-bearing spermatozoa with a first zinc finger protein conjugated, complexed, or fused with a first toxic sequence enabled reassembly (SEER) marker, wherein said first zinc finger protein is specific for either the population of X chromosome-bearing spermatozoa or Y chromosome-bearing spermatozoa;
- (b) contacting the mammalian sperm sample comprising a population of X chromosome-bearing spermatozoa and a population of Y chromosome-bearing spermatozoa with a second zinc finger protein conjugated, complexed, or fused a second toxic sequence enabled reassembly (SEER) marker, wherein said second zinc finger is specific for either the population of X chromosome-bearing spermatozoa or Y chromosome-bearing spermatozoa;
- (c) subjecting the mammalian sperm cells to conditions which allow for reassembly to occur between said first toxic sequence enabled reassembly (SEER) marker and said toxic second sequence enabled reassembly (SEER) marker;
- and
- (d) recovering the unbound sperm cells.
2 more elements
Dependencies are inferred from the claim text.