WO2005043991A1 - Methods for selecting gametes and for producing genetically modified non-human animals - Google Patents

Methods for selecting gametes and for producing genetically modified non-human animals Download PDF

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Publication number
WO2005043991A1
WO2005043991A1 PCT/GB2004/050021 GB2004050021W WO2005043991A1 WO 2005043991 A1 WO2005043991 A1 WO 2005043991A1 GB 2004050021 W GB2004050021 W GB 2004050021W WO 2005043991 A1 WO2005043991 A1 WO 2005043991A1
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selectable marker
genetic modification
sperm
gametes
genetically modified
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PCT/GB2004/050021
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French (fr)
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John C. Saunders
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Babraham Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • This invention relates to methods for selection of gametes from chimeric non- human animals, in particular for selection of genetically modified sperm or ova from chimeric non-human animals. This invention further relates to improved methods for producing genetically modified non-human animals and methods for improving the efficiency of germline transmission of genetic modifications in non-human animals.
  • genetic material is altered from its natural state; the alteration can involve introduction of foreign DNA into the genome, deletion of native DNA from the genome, or substitution of foreign DNA for native DNA.
  • Transgenic animals are genetically modified so that they contain artificially introduced genes inserted or substituted into the genome.
  • the introduced gene or genes can confer a new function ("knock-in"), e.g. so that the modified animal encodes a new protein.
  • the introduced genetic material may interrupt or alter the coding sequence of a native gene, or a segment of native gene may be deleted, both strategies can result in loss of gene function, in which instance the animal is termed a "knock-ouf animal.
  • Geneticaily modified animals are useful for studying gene function and regulation, allowing investigation of the impact of a genetic change on the whole organism. Genetically modified animals are used as model systems to investigate human diseases and conditions related to genetic disruption, such as overexpression, incorrect expression or absence of a defined protein. Artificially introduced genetic material is commonly called “foreign" or exogenous DNA, it may carry one or more gene(s) and may be derived from any source. In animals with full genetic modification, the foreign genetic material is integrated into the genome in every cell of that animal.
  • Techniques for genetic manipulation have been developed in animals such as rodents, in particular mice.
  • foreign DNA is introduced into an early embryo.
  • Commonly used methods include pronuclear microinjection and the introduction of DNA into embryonic stem cells.
  • foreign DNA is introduced into a fertilised egg.
  • the foreign DNA is injected into a nucleus in the fertilised egg, usually the large male pronucleus derived from sperm.
  • foreign DNA integrates at random positions in the genome, usually as multiple tandem copies of the DNA.
  • the resulting animal is genetically modified, and, provided that the genetic modification has occurred in the germline (the cell line from which the gametes are generated), the modification can be passed to subsequent generations, so animals bred from the sperm or ova will also be genetically modified.
  • Pronuclear injection is a technically difficult, costly technique, which is impractical in many animal species, thus alternative methods have been developed.
  • ES cells are derived from very early embryos and are pluripotent, i.e> they can differentiate Into multiple cell types when introduced into another early embryo, such as a blastocyst
  • the DNA introduced into embryonic stem cells may integrate randomly, or a targeted integration method may be used.
  • part of the introduced DNA is homologous, i.e. identical or very similar to that of the host cell genome, allowing homologous recombination to occur, so that the foreign DNA is introduced at a specific site of homoiogy.
  • the transformed embryonic stem cells containing foreign DNA are then introduced into an early embryo, e.g. a blastocyst, which is implanted into a female host if introduction of the foreign DNA has been successful, then the embryo will develop into a chimeric animal.
  • the introduced DNA may contribute to the germline of the chimeric animal. If genetically modified cells contribute to the germline, the cell line from which egg or sperm celts (gametes) are generated, then in male chimera some transgenic sperm will be produced, in female chimera some transgenic eggs (ova) will be produced, but these will be mixed with non-transgenic sperm or ova respectively.
  • a mixture of sperm is produced from either germline cells derived from the blastocyst, or germline cells derived from the transformed ES cells, the latter having the genetic modification.
  • the mixture of sperm derived from the blastocyst cells and modified sperm derived from the ES cell can be used to fertilise a normal egg, to produce either a wild type or a genetically modified mouse respectively.
  • Retroviruses are capable of stable integration into the genome.
  • silencing of oncoretroviruses during development has been found to result in very low levels of transgene expression.
  • Lois et al. Science (2002), 295, 868-872 have described an alternative retroviral approach to generate transgenic mice, in this method, lentiviruses are used as transgene delivery systems, which can infect dividing and non-dividing ceils resulting in production of chimeric mice. These chimeric animals are bred to produce progeny such as transgenic "knock-in" mice that express high levels of foreign protein.
  • a problem with the various techniques is that the chimeric progeny obtained following gene introduction must be bred in order to ensure germline transmission. Often successive matings are required. The process is time consuming and results in high costs as many animals must be bred to obtain germline transgenic animals. In some instances germline transmission is never achieved.
  • the methods of the invention not only reduce the costs associated with animal breeding, but can achieve germline transmission of a genetic modification at the first attempt.
  • the present invention provides a method for selecting, i.e. identifying and/or sorting, isolating or separating geneticaily modified gametes, i.e. genetically modified sperm or ova.
  • the present invention provides a method for selecting a genetically modified gamete obtained from a chimeric non-human animal producing heterogeneous gametes, characterised by sorting gametes having a first genetic modification enabling expression of a first selectable marker from gametes that do not express the first selectable marker.
  • the invention further provides a method for selecting a genetically modified non-human animal gamete comprising: (a) providing a chimeric non-human animal producing heterogeneous gametes, said gametes:
  • selection methods of the invention can be used to sort heterogeneous gametes that have a first genetic modification to enable expression of a first selectable marker from those that have no genetic modification.
  • selection methods of the invention may be used to separate gametes that have a first genetic modification to enable expression of a first selectable marker from those that have a second genetic modification.
  • Gametes are sorted by means of the selectable marker into gametes that express the first selectable marker and gametes that do not express the selectable marker.
  • a second genetic modification may enable expression of a second selectable marker; in which instance the gametes can be sorted by means of the first and/or second selectable marker, the first and second selectable markers being different markers.
  • the invention also provides a genetically modified gamete obtained using a selection method according to the invention.
  • the present invention provides a method for producing a genetically modified non-human animal comprising selecting a genetically modified non-human gamete using a selection method of the invention and using the gamete to generate a genetically modified non-human animal, for example by a method such as IVF or ICSI.
  • the invention provides a method for producing a genetically modified non-human animal comprising
  • the second genetic modification does not enable expression of a selectable marker and gametes are sorted on the basis of the first marker, into gametes expressing the first selectable marker and gametes having the second genetic modification.
  • the gamete can be a sperm or an ovum.
  • the first genetic modification is suitably an insertion of exogenous (foreign) DNA comprising a selectable marker or a substitution (replacement) of endogenous DNA with exogenous DNA comprising a selectable marker.
  • the exogenous DNA may further comprise one or more exogenous gene(s) and the one or more exogenous gene(s) may be operabiy linked to the selectable marker.
  • the second genetic modification can be an insertion of exogenous (foreign) DNA, a substitution (replacement) of endogenous DNA with exogenous DNA, or a deletion of endogenous DNA.
  • the second genetic modification can be an insertion of exogenous DNA comprising a second selectable marker or a substitution (replacement) of endogenous DNA with exogenous DNA comprising a second selectable marker.
  • the exogenous DNA may further comprise one or more exogenous gene(s), that may be operabiy linked to said second selectable marker for expression.
  • foreign (exogenous) DNA can be introduced into a pluripotent cell such as a stem cell, more preferably an embryonic stem (ES) cell, which is then introduced into an early embryo, such as a blastocyst.
  • a pluripotent cell such as a stem cell, more preferably an embryonic stem (ES) cell
  • ES embryonic stem
  • the pluripotent cell is preferably a stem cell, more preferably an embryonic stem ceil.
  • the early embryo is preferably a blastocyst.
  • the invention provides a method for producing a genetically modified non-human animal comprising:
  • a selectable marker used in a method of the invention is a fluorescent marker or is labelled with a fluorescent moiety.
  • the first and/or second selectable marker(s) is preferably a fluorescent protein.
  • a fluorescent protein or proteins appropriate for use in methods of the invention can be selected from the group comprising a red, orange, yellow, yellow-green, green-yellow, green or cyan fluorescent protein.
  • the first or second selectable marker is a green fluorescent protein.
  • the first and/or second fluorescent protein can be a wild type, enhanced, destabilised enhanced, or red-shift fluorescent protein.
  • the selectable marker is preferably operatively linked to a promoter to enable expression of the selectable marker.
  • Expression may be constitutive or induced, but is preferably constitutive.
  • Expression of the selectable marker may be cell specific, where the selectable marker is operatively linked to a promoter capable of expression in specific cell types, e.g. germline cells, gametes.
  • the sperm itself is not fluorescent, but can be labelled prior to sorting with a fluorescent marker.
  • the first and/or second selectable marker can be a surface antigen specifically labelled, directly or indirectly, with a fluorescent moiety.
  • the fluorescent moiety can be a fluorescent labelled molecule, preferably a fluorescent labelled antibody or a fluorescent labelled antigen binding fragment of an antibody, or a fluorescent labelled antigen binding fragment of a protein.
  • gametes are selected by sorting the gametes into fluorescent and non-fluorescent samples. This can be done by hand or by an automated method, such as FACS analysis. Hand sorting is preferred for ova, which are generally too delicate to be sorted by traditional automated methods. In contrast an automated method such as FACS analysis is preferred for sperm. Sorting sperm into fluorescent and non-fluorescent pools enables selection of the transgenic sperm.
  • the gametes are a heterogeneous mixture, comprising one type of gametes that express a first selectable marker and a further type of gametes which express a second, different, selectable marker
  • the gametes can be sorted by means of the first and/or second selectable marker.
  • the first and second selectable markers are fluorescent markers that fluoresce at different wavelengths
  • gametes of interest can be selected by sorting gametes into samples that fluoresce at different wavelengths.
  • Methods of the invention are applicable to many non-human animals and are, for example, suitable when the genetically modified non-human animal is a horse, cow, primate (such as a lemur, tarsier, monkey or ape), sheep, goat, pig, dog, cat, chicken, rabbit, fish, or a rodent, such as a guinea pig, rat or mouse.
  • Methods of the invention are particularly useful when the genetically modified non human animal is a rodent, preferably a rat or mouse.
  • the invention provides a genetically modified non-human animal obtainable or obtained according to a method for producing a genetically modified non-human animal according to the invention. Also provided is the use, in the production of a genetically modified non-human animal, of a genetically modified gamete obtained using a selection method of the invention.
  • non-human animals such as rodents, in particular rats or mice
  • rodents in particular rats or mice
  • a fluorescent protein using a lentiviral approach such as that described by Lois ef a/. Science (2002), 295, 868-872.
  • Biastocysts from these animals are capable of expressing fluorescent protein and can be used in methods of the invention as host embryos into which foreign DNA is introduced by introduction of a genetically modified ES cell, e.g. transformed with foreign DNA so that it harbours a transgene or transgenes.
  • the present invention provides a method for producing a genetically modified rodent, comprising:
  • the selectable marker is a fluorescent protein, more preferably a green fluorescent protein.
  • the Invention provides a method for producing a genetically modified mouse, comprising: (a) providing a mouse blastocyst having a first genetic modification to enable expression of a green fluorescent protein,
  • the early embryo background can be non- fluorescent and the genetic modification in the pluripotent cell can be introduction of exogenous DNA that comprises a selectable marker, in particular a fluorescent selectable marker.
  • the introduced DNA which may be inserted or substituted into the pluripotent cell genome, may comprise a gene encoding a fluorescent protein, which may be linked to a further transgene or transgenes.
  • Methods in which the genetically modified sperm are fluorescent are useful for species where the sperm tail is "short" thus allowing 100% separation, but are less effective for separating long tailed sperm, from species such as mice.
  • blastocysts from mice expressing a fluorescent protein are used as recipients for embryonic stem cells from a different strain containing altered
  • DNA (e.g. targeted ES cells, harbouring foreign DNA).
  • ES-blastocysts are implanted into a female host and chimeric progeny are produced.
  • the sperm of the male chimera are a mixture of fluorescent sperm, e.g. derived from a GFP mouse blastocyst, and non-fluorescent sperm having the genetic modification of interest, the latter sperm being derived from the genetically modified ES cells.
  • the sperm is sorted into fluorescent and non-fluorescent samples, either by hand, or preferably by an automated method, for example FACS analysis. This method is particularly useful for enrichment of transgenic sperm from mice, because mouse sperm has a very long tail and tends to intertwine, making it difficult to achieve effective separation.
  • Sperm which are not fluorescing i.e. which are derived from the genetically modified ES cell, are used to generate a mouse germline genetic modification, e.g. by either IVF or ICSI.
  • the present invention provides a method for producing a transgenic mouse, comprising:
  • transgenic progeny e.g. by IVF or ICSI.
  • the first selectable marker is a fluorescent marker, such as a fluorescent protein, or is labelled with a fluorescent marker.
  • the transgene of interest is within the non-fluorescing sperm pool. In this situation, when the sperm are sorted, it does not matter if the fluorescent sperm pool contains non- fluorescing sperm, as the fluorescent sperm is not the transgenic sperm of interest and is not used for generation of progeny. In the non-fluorescing pool, containing the transgenic sperm, any fluorescing sperm that have not been removed can be readily identified, It is thus possible to determine the degree of enrichment of transgenic sperm in the sample and re-sort if desired to achieve further enrichment. Absolute separation of fluorescing and non-fluorescing sperm is desirable, but not essential, selective enrichment of non-fluorescing transgenic sperm within the sample is sufficient to improve the efficiency of germline transmission.
  • Gametes selected for presence of the genetic modification are used to generate genetically modified progeny with a genetically altered germline.
  • Methods to produce progeny include iCSI (intra-cytopla ⁇ mic sperm injection) and IVF (in vitro fertilisation).
  • ICSI methods are preferred over IVF because "dead" sperm can be used in ICSI methods, thus any problems of sperm deterioration, e.g. on storage or during handling or sorting, are alleviated.
  • the ICSI method can be used in a wider range of strains than IVF, which does not work satisfactorily for some transgenic animals. For example, IVF does not work efficiently in mice of the C57BI/6 background which is the predominantly used mouse model.
  • An advantage of methods of the invention is that only one low percentage chimera is needed to enable germline transmission. Methods of the invention result in lower costs compared to current methods, as the numbers of animals needed to obtain germline transmission is reduced.
  • the probability of achieving germline transmission is highly dependent on the ES cell line used. Certain ES ceil lines are known to exhibit poor germline capabilities. Even with a "good" ES cell line, using traditional methods, a large number of chimeras must be produced in order to increase the likelihood of achieving germline transmission. A succession of pregnancies must be completed before the correct sperm or ova come through in the germline. Using methods of the invention, efficient germline transmission can be achieved using any ES cell line.
  • transgenic gametes i.e. sperm or ova
  • germline transmission is achieved with high efficiency, regardless of the cell line used or quality of chimera.
  • efficient germline transmission can be achieved from both poor ES cells and low percentage chimeras.
  • a DNA construct is made containing a ubiquitous promoter coupled to a fluorescent protein, enabling this protein to be expressed in all tissues, but most importantly in the germ cells.
  • blastocysts Targeted embryonic stem cells from a different strain of mouse or the same strain, into which foreign DNA has been introduced, are injected into blastocysts.
  • the injected blastocysts are implanted into a recipient female mouse and develop into chimeric progeny *
  • a chimeric progeny pup has two types of germ cells, derived either from the fluorescent protein donor mouse blastocyst or from the targeted embryonic stem cells.
  • Sperm is collected from a male chimera, the quality of the chimera can be of a very low percentage.
  • the sperm of the chimera are sorted by FACS and those sperm not fluorescing, i.e. those with the transgene rather than the fluorescent marker, are used to generate germline transmission by either IVF or ICSI.
  • the methods should be carried out by competent personnel, proficient in mouse embryology to such a standard as to be proficient in embryo handling, ES cell injection and IVF in the mouse.
  • FACS sorting can be carried out by different personnel and this facility is available commercially.
  • Transgenic blastocysts are used to create knockout mice via ES cell injection.
  • the strain of mice from which the blastocysts are derived C57BL 6 TG (ACTBEGFP) 1 OSB/J, stock# 003291 (JAX Mouse Lab, USA), has a ubiquitous green fluorescent protein (GFP) expressed in ail cells.
  • Green fluorescent blastocysts are obtained and injected with non fluorescing ES cells to create a mosaic mouse (chimera) in which the blastocyst derived sperm cells express green fluorescent protein and the genetically modified sperm, derived from the ES cell, do not produce green fluorescent protein.
  • GFP mouse day 4 embryos are produced by mating two GFP mice together and looking for the presence of a vaginal plug to indicate that mating has taken place.
  • the uterus is dissected and placed in saline or FHM medium (Methods in Enzymology 225:153-164) at room temperature.
  • Embryos are flushed out of the uterus with FHM media and collected in a Petri dish. They are then harvested from the Petri dish using small glass pipettes and placed into drops of FHM under paraffin oil (Sigma: M8410).
  • the blastocysts are then placed in an incubator at 37°C or left on the bench at room temperature for 1 hour until use.
  • ES cells are transfected with foreign DNA of known sequence having a marker for selection and designed to alter a target gene of interest After homologous recombination, correctly targeted clones are expanded for injection into the GFP blastocysts.
  • GFP blastocysts with large blastocele cavities are selected and the transformed .
  • ES cells are injected into the cavity. This is carried out using a micro- manipulation system that allows precise manipulation to be carried out, resulting in an embryo which is a GFP blastocyst with non-GFP ES cells within its blastocel. Typically 6-20 cells are placed in each blastocyst. These embryos are then placed (in a group of 10-12 embryos) into the uterus of a pseudopregnant recipient mouse. Pseudopregnancy is induced by allowing a female mouse to mate with an infertile (vasectomised) male mouse.
  • the recipient mouse is anaesthetised and the uterus withdrawn from the body.
  • the embryos are placed into the uterus via a small glass needle through a small puncture hole in the side of the uterus.
  • the embryos are drawn up the lumen of the needle and placed into the lumen of the uterus.
  • the needle is withdrawn and the uterus replaced into the body cavity.
  • the skin is closed with suture clips and the mouse is allowed to recover.
  • Chimeric mice After a gestation of 3 weeks the pups are delivered. Chimeric mice can be identified as they are black/agouti mosaics, because the GFP mouse strain from which the blastocyst ceils are derived has black fur and the mouse strain from which the ES cells are derived have agouti fur. Male chimeras produce two populations of sperm: one population being derived from the black (GFP) mouse cells and the other derived from the ES cell having the genetic modification of interest.
  • GFP black
  • ES cell having the genetic modification of interest Male chimeras produce two populations of sperm: one population being derived from the black (GFP) mouse cells and the other derived from the ES cell having the genetic modification of interest.
  • the chimeric mouse would be paired to a number of females and the pups analysed, e.g. by PCR or Southern blotting, to see if transgenic offspring have been made; confirmation that a mouse had failed to produce transgenic sperm could be obtained only after exhaustive pairings.
  • sperm from male chimera is collected and examined. Chimeric sperm is recovered from the chimera by culling the male chimeric animal and removing one or both vas deferens.
  • the mouse can be anaesthetised and an operation performed to remove one vas deferens. Sperm is squeezed from the vas deferens and allowed to swim into a small amount of HTF fertilization media (Quinn. (1985) Fertility and Sterility, 44; 493-498).
  • sperm that do not carry the desired genetic modification are derived from the GFP mouse cell and will fluoresce green after exposure to light at an appropriate excitation wavelength.
  • Sperm carrying the desired genetic modification (derived from the ES cell) is not fluorescent on exposure to light at the excitation wavelength.
  • Hand sorting or FACS sorting is used to separate the fluorescent and non- fluorescent sperm to isolate the non-fluorescent sperm having the desired genetic modification.
  • the incubated embryos are eventually implanted, at 2 cell or blastocyst stage, into a pseudopregnant recipient donor mother (as described above) and the genetically modified embryos are grown to birth.
  • the transgenic mice produced are the founders of a unique line of mice.

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Abstract

The present invention relates to methods for producing genetically modified animals comprising: a) providing a blastocyst having a first genetic modification to enable expression of selectable marker, b) introducing into said blastocyst an ES cell having second genetic modification, c) implanting the ES-blastocyst into a female host and generating chimeric male progeny, d) collecting sperm from said male chimeric progeny, e) sorting sperm by means of selectable marker into sperm expressing the selectable marker and sperm having the second genetic modification, and f) using sperm having the second genetic modification to generate a genetically modified animal.

Description

M&C Folio: WPP290040
Methods for selecting gametes and for producing genetically modified non-human animals
This invention relates to methods for selection of gametes from chimeric non- human animals, in particular for selection of genetically modified sperm or ova from chimeric non-human animals. This invention further relates to improved methods for producing genetically modified non-human animals and methods for improving the efficiency of germline transmission of genetic modifications in non-human animals.
Background
In the cells of genetically modified animals, genetic material is altered from its natural state; the alteration can involve introduction of foreign DNA into the genome, deletion of native DNA from the genome, or substitution of foreign DNA for native DNA. Transgenic animals are genetically modified so that they contain artificially introduced genes inserted or substituted into the genome. The introduced gene or genes can confer a new function ("knock-in"), e.g. so that the modified animal encodes a new protein. The introduced genetic material may interrupt or alter the coding sequence of a native gene, or a segment of native gene may be deleted, both strategies can result in loss of gene function, in which instance the animal is termed a "knock-ouf animal. Geneticaily modified animals are useful for studying gene function and regulation, allowing investigation of the impact of a genetic change on the whole organism. Genetically modified animals are used as model systems to investigate human diseases and conditions related to genetic disruption, such as overexpression, incorrect expression or absence of a defined protein. Artificially introduced genetic material is commonly called "foreign" or exogenous DNA, it may carry one or more gene(s) and may be derived from any source. In animals with full genetic modification, the foreign genetic material is integrated into the genome in every cell of that animal.
Techniques for genetic manipulation have been developed in animals such as rodents, in particular mice. To obtain genetically modified animals, foreign DNA is introduced into an early embryo. Commonly used methods include pronuclear microinjection and the introduction of DNA into embryonic stem cells.
In pronuclear injection, foreign DNA is introduced into a fertilised egg. The foreign DNA is injected into a nucleus in the fertilised egg, usually the large male pronucleus derived from sperm. Generally, foreign DNA integrates at random positions in the genome, usually as multiple tandem copies of the DNA. The resulting animal is genetically modified, and, provided that the genetic modification has occurred in the germline (the cell line from which the gametes are generated), the modification can be passed to subsequent generations, so animals bred from the sperm or ova will also be genetically modified.
Pronuclear injection is a technically difficult, costly technique, which is impractical in many animal species, thus alternative methods have been developed.
In a second method, foreign DNA is introduced into an embryonic stem (ES) cell. ES cells are derived from very early embryos and are pluripotent, i.e> they can differentiate Into multiple cell types when introduced into another early embryo, such as a blastocyst The DNA introduced into embryonic stem cells may integrate randomly, or a targeted integration method may be used. In targeted integration methods, part of the introduced DNA is homologous, i.e. identical or very similar to that of the host cell genome, allowing homologous recombination to occur, so that the foreign DNA is introduced at a specific site of homoiogy.
The transformed embryonic stem cells containing foreign DNA are then introduced into an early embryo, e.g. a blastocyst, which is implanted into a female host if introduction of the foreign DNA has been successful, then the embryo will develop into a chimeric animal. The introduced DNA may contribute to the germline of the chimeric animal. If genetically modified cells contribute to the germline, the cell line from which egg or sperm celts (gametes) are generated, then in male chimera some transgenic sperm will be produced, in female chimera some transgenic eggs (ova) will be produced, but these will be mixed with non-transgenic sperm or ova respectively. In male chimeric offspring, a mixture of sperm is produced from either germline cells derived from the blastocyst, or germline cells derived from the transformed ES cells, the latter having the genetic modification. The mixture of sperm derived from the blastocyst cells and modified sperm derived from the ES cell, can be used to fertilise a normal egg, to produce either a wild type or a genetically modified mouse respectively.
As an alternative approach, foreign DNA has been introduced into ceils using retroviruses developed as gene delivery vehicles. Retroviruses are capable of stable integration into the genome. However, in some instances, silencing of oncoretroviruses during development has been found to result in very low levels of transgene expression. Lois et al. Science (2002), 295, 868-872 have described an alternative retroviral approach to generate transgenic mice, in this method, lentiviruses are used as transgene delivery systems, which can infect dividing and non-dividing ceils resulting in production of chimeric mice. These chimeric animals are bred to produce progeny such as transgenic "knock-in" mice that express high levels of foreign protein.
A problem with the various techniques is that the chimeric progeny obtained following gene introduction must be bred in order to ensure germline transmission. Often successive matings are required. The process is time consuming and results in high costs as many animals must be bred to obtain germline transgenic animals. In some instances germline transmission is never achieved.
The methods of the invention not only reduce the costs associated with animal breeding, but can achieve germline transmission of a genetic modification at the first attempt.
Disclosure of Invention
Broadly, the present invention provides a method for selecting, i.e. identifying and/or sorting, isolating or separating geneticaily modified gametes, i.e. genetically modified sperm or ova.
The present invention provides a method for selecting a genetically modified gamete obtained from a chimeric non-human animal producing heterogeneous gametes, characterised by sorting gametes having a first genetic modification enabling expression of a first selectable marker from gametes that do not express the first selectable marker.
The invention further provides a method for selecting a genetically modified non-human animal gamete comprising: (a) providing a chimeric non-human animal producing heterogeneous gametes, said gametes:
(i) having a first genetic modification enabling expression of a first selectable marker, or
(ii) not expressing the first selectable marker; (b) collecting gametes from said chimeric non-human animal, and sorting gametes having a first genetic modification enabling expression of a first selectable marker from gametes that do not express the first selectable marker. Selection methods of the invention can be used to sort heterogeneous gametes that have a first genetic modification to enable expression of a first selectable marker from those that have no genetic modification. Alternatively, selection methods of the invention may be used to separate gametes that have a first genetic modification to enable expression of a first selectable marker from those that have a second genetic modification. Gametes are sorted by means of the selectable marker into gametes that express the first selectable marker and gametes that do not express the selectable marker. In certain embodiments of the invention, a second genetic modification may enable expression of a second selectable marker; in which instance the gametes can be sorted by means of the first and/or second selectable marker, the first and second selectable markers being different markers.
These methods for selecting genetically modified gametes are useful for selective enrichment of geneticaily modified gametes of interest from a mixed sample. The invention also provides a genetically modified gamete obtained using a selection method according to the invention.
The present invention provides a method for producing a genetically modified non-human animal comprising selecting a genetically modified non-human gamete using a selection method of the invention and using the gamete to generate a genetically modified non-human animal, for example by a method such as IVF or ICSI.
Further provided is a method for producing a genetically modified non-human animal comprising:
(a) providing an early embryo having a first genetic modification to enable expression of a first selectable marker, (b) introducing into said early embryo a pluripotent cell:
(i) having no genetic modification; or,
(it) having a second genetic modification; or, (iii) having a second genetic modification to enable expression of a second selectable marker;
(c) implanting the early embryo obtained in (b) into a female host to obtain chimeric progeny, (d) collecting gametes from chimeric progeny obtained in (c),
(e) sorting gametes by means of a or the selectable marker(s), and,
(f) using selected gametes to generate a genetically modified non-human animal, preferably by IVF or ICSI.
In a preferred aspect, the invention provides a method for producing a genetically modified non-human animal comprising
(a) providing an early embryo having a first genetic modification to enable expression of a first selectable marker,
(b) introducing into said early embryo a pluripotent cell having a second genetic modification,
(c) implanting the early embryo obtained in (b) into a female host to obtain chimeric progeny,
(d) collecting gametes from chimeric progeny obtained in (c),
(e) sorting gametes by means of the first selectable marker, and, (f) using a gamete having the second genetic modification to generate a genetically modified non-human animal, preferably by IVF or ICSI.
In a particularly preferred embodiment, the second genetic modification does not enable expression of a selectable marker and gametes are sorted on the basis of the first marker, into gametes expressing the first selectable marker and gametes having the second genetic modification.
In methods of the invention, the gamete can be a sperm or an ovum.
In methods of the invention, the first genetic modification is suitably an insertion of exogenous (foreign) DNA comprising a selectable marker or a substitution (replacement) of endogenous DNA with exogenous DNA comprising a selectable marker. The exogenous DNA may further comprise one or more exogenous gene(s) and the one or more exogenous gene(s) may be operabiy linked to the selectable marker.
When present, the second genetic modification can be an insertion of exogenous (foreign) DNA, a substitution (replacement) of endogenous DNA with exogenous DNA, or a deletion of endogenous DNA. The second genetic modification can be an insertion of exogenous DNA comprising a second selectable marker or a substitution (replacement) of endogenous DNA with exogenous DNA comprising a second selectable marker. The exogenous DNA may further comprise one or more exogenous gene(s), that may be operabiy linked to said second selectable marker for expression.
To generate a chimeric animal capable of producing heterogeneous gametes, foreign (exogenous) DNA can be introduced into a pluripotent cell such as a stem cell, more preferably an embryonic stem (ES) cell, which is then introduced into an early embryo, such as a blastocyst. The pluripotent cell is preferably a stem cell, more preferably an embryonic stem ceil. The early embryo is preferably a blastocyst.
The invention provides a method for producing a genetically modified non- human animal comprising:
(a) providing a blastocyst having a first genetic modification to enable expression of a first selectable marker, (b) introducing into said blastocyst an ES ceil having a second genetic modification,
(c) implanting the ES-blastocyst into a female host and generating chimeric progeny,
(d) collecting sperm from male chimeric progeny, (e) sorting sperm by means of the selectable marker into sperm expressing the first selectable marker and sperm having the second genetic modification, and, (f) using sperm having the second genetic modification to generate geneticalfy modified progeny, preferably by IVF or ICSI.
Suitably a selectable marker used in a method of the invention is a fluorescent marker or is labelled with a fluorescent moiety. In methods involving expression of first or second selectable markers by genotypically different gametes, the first and second selectable markers differ from each other. The first and/or second selectable marker(s) is preferably a fluorescent protein. A fluorescent protein or proteins appropriate for use in methods of the invention can be selected from the group comprising a red, orange, yellow, yellow-green, green-yellow, green or cyan fluorescent protein. In a particularly preferred embodiment of the invention the first or second selectable marker is a green fluorescent protein. The first and/or second fluorescent protein can be a wild type, enhanced, destabilised enhanced, or red-shift fluorescent protein.
The selectable marker is preferably operatively linked to a promoter to enable expression of the selectable marker. Expression may be constitutive or induced, but is preferably constitutive. Expression of the selectable marker may be cell specific, where the selectable marker is operatively linked to a promoter capable of expression in specific cell types, e.g. germline cells, gametes.
In certain embodiments of the invention, the sperm itself is not fluorescent, but can be labelled prior to sorting with a fluorescent marker. The first and/or second selectable marker can be a surface antigen specifically labelled, directly or indirectly, with a fluorescent moiety. The fluorescent moiety can be a fluorescent labelled molecule, preferably a fluorescent labelled antibody or a fluorescent labelled antigen binding fragment of an antibody, or a fluorescent labelled antigen binding fragment of a protein.
Where the gametes are a heterogeneous mixture of fluorescent and non- fluorescent gametes, gametes are selected by sorting the gametes into fluorescent and non-fluorescent samples. This can be done by hand or by an automated method, such as FACS analysis. Hand sorting is preferred for ova, which are generally too delicate to be sorted by traditional automated methods. In contrast an automated method such as FACS analysis is preferred for sperm. Sorting sperm into fluorescent and non-fluorescent pools enables selection of the transgenic sperm.
If the gametes are a heterogeneous mixture, comprising one type of gametes that express a first selectable marker and a further type of gametes which express a second, different, selectable marker, then the gametes can be sorted by means of the first and/or second selectable marker. For example, where the first and second selectable markers are fluorescent markers that fluoresce at different wavelengths, gametes of interest can be selected by sorting gametes into samples that fluoresce at different wavelengths.
Methods of the invention are applicable to many non-human animals and are, for example, suitable when the genetically modified non-human animal is a horse, cow, primate (such as a lemur, tarsier, monkey or ape), sheep, goat, pig, dog, cat, chicken, rabbit, fish, or a rodent, such as a guinea pig, rat or mouse. Methods of the invention are particularly useful when the genetically modified non human animal is a rodent, preferably a rat or mouse.
The invention provides a genetically modified non-human animal obtainable or obtained according to a method for producing a genetically modified non-human animal according to the invention. Also provided is the use, in the production of a genetically modified non-human animal, of a genetically modified gamete obtained using a selection method of the invention.
So that methods of the invention may be performed, non-human animals such as rodents, in particular rats or mice, can be engineered to express a fluorescent protein using a lentiviral approach such as that described by Lois ef a/. Science (2002), 295, 868-872. Biastocysts from these animals are capable of expressing fluorescent protein and can be used in methods of the invention as host embryos into which foreign DNA is introduced by introduction of a genetically modified ES cell, e.g. transformed with foreign DNA so that it harbours a transgene or transgenes.
Accordingly, the present invention provides a method for producing a genetically modified rodent, comprising:
(a) providing a blastocyst having a first genetic modification to enable expression of a selectable marker,
(b) introducing into said blastocyst an ES cell having a second genetic modification,
(c) implanting the ES-blastocyst Into a female host and generating a chimeric male progeny rodent,
(d) collecting sperm from said male chimeric progeny rodent,
(e) sorting sperm by means of the selectable marker into sperm expressing the selectable marker and sperm having the second genetic modification, and,
(e) using sperm having the second genetic modification to generate a genetically modified progeny rodent, preferably by IVF or ICSI.
In a preferred embodiment of this method for producing a genetically modified rodent, the selectable marker is a fluorescent protein, more preferably a green fluorescent protein.
The Invention provides a method for producing a genetically modified mouse, comprising: (a) providing a mouse blastocyst having a first genetic modification to enable expression of a green fluorescent protein,
(b) introducing into said blastocyst a mouse ES cell having a second genetic modification,
(c) implanting the ES-blastocyst into a female mouse and generating a chimeric male progeny mouse,
(d) collecting sperm from said male chimeric progeny mouse, (e) sorting sperm by means of the selectable marker into sperm expressing the selectable marker and sperm having the second genetic modification, and,
(f) using sperm having the second genetic modification to generate genetically modified progeny, preferably by IVF or ICSI.
In alternative embodiments of methods for the production of genetically modified non-human animals, the early embryo background can be non- fluorescent and the genetic modification in the pluripotent cell can be introduction of exogenous DNA that comprises a selectable marker, in particular a fluorescent selectable marker. For example the introduced DNA, which may be inserted or substituted into the pluripotent cell genome, may comprise a gene encoding a fluorescent protein, which may be linked to a further transgene or transgenes.
Methods in which the genetically modified sperm are fluorescent are useful for species where the sperm tail is "short" thus allowing 100% separation, but are less effective for separating long tailed sperm, from species such as mice.
In preferred methods of the invention, blastocysts from mice expressing a fluorescent protein, preferably a green fluorescent protein, are used as recipients for embryonic stem cells from a different strain containing altered
DNA, (e.g. targeted ES cells, harbouring foreign DNA). ES-blastocysts are implanted into a female host and chimeric progeny are produced. The sperm of the male chimera are a mixture of fluorescent sperm, e.g. derived from a GFP mouse blastocyst, and non-fluorescent sperm having the genetic modification of interest, the latter sperm being derived from the genetically modified ES cells.
The sperm is sorted into fluorescent and non-fluorescent samples, either by hand, or preferably by an automated method, for example FACS analysis. This method is particularly useful for enrichment of transgenic sperm from mice, because mouse sperm has a very long tail and tends to intertwine, making it difficult to achieve effective separation. Sperm which are not fluorescing, i.e. which are derived from the genetically modified ES cell, are used to generate a mouse germline genetic modification, e.g. by either IVF or ICSI.
The present invention provides a method for producing a transgenic mouse, comprising:
(a) introducing a transgenic mouse ES cell into a mouse blastocyst capable of expressing a first selectable marker,
(b) implanting the ES-blastocyst into a compatible female mouse and generating a chimeric male progeny mouse, (c) collecting sperm from said male chimeric progeny mouse,
(d) sorting sperm by means of the first selectable marker into sperm expressing selectable marker and sperm having the transgene or transgenes, and,
(e) using sperm having the transgene or transgenes to generate transgenic progeny, e.g. by IVF or ICSI.
In a preferred method the first selectable marker is a fluorescent marker, such as a fluorescent protein, or is labelled with a fluorescent marker. The transgene of interest is within the non-fluorescing sperm pool. In this situation, when the sperm are sorted, it does not matter if the fluorescent sperm pool contains non- fluorescing sperm, as the fluorescent sperm is not the transgenic sperm of interest and is not used for generation of progeny. In the non-fluorescing pool, containing the transgenic sperm, any fluorescing sperm that have not been removed can be readily identified, It is thus possible to determine the degree of enrichment of transgenic sperm in the sample and re-sort if desired to achieve further enrichment. Absolute separation of fluorescing and non-fluorescing sperm is desirable, but not essential, selective enrichment of non-fluorescing transgenic sperm within the sample is sufficient to improve the efficiency of germline transmission.
Gametes selected for presence of the genetic modification (e.g. introduction of a transgene or transgenes) are used to generate genetically modified progeny with a genetically altered germline. Methods to produce progeny include iCSI (intra-cytoplaεmic sperm injection) and IVF (in vitro fertilisation). ICSI methods are preferred over IVF because "dead" sperm can be used in ICSI methods, thus any problems of sperm deterioration, e.g. on storage or during handling or sorting, are alleviated. Furthermore, the ICSI method can be used in a wider range of strains than IVF, which does not work satisfactorily for some transgenic animals. For example, IVF does not work efficiently in mice of the C57BI/6 background which is the predominantly used mouse model.
An advantage of methods of the invention is that only one low percentage chimera is needed to enable germline transmission. Methods of the invention result in lower costs compared to current methods, as the numbers of animals needed to obtain germline transmission is reduced.
Using currently available methods, germline transmission is not readily achievable, if at all, with low percentage chimeras. Techniques of the invention overcome this by permitting selection of genetically modified gametes, in particular genetically modified sperm from low percentage chimeras. Methods of the invention do not require the use of large numbers of animals and blastocysts and provide successful germline transmission in a reduced time scale.
For methods involving ES ceils, the probability of achieving germline transmission is highly dependent on the ES cell line used. Certain ES ceil lines are known to exhibit poor germline capabilities. Even with a "good" ES cell line, using traditional methods, a large number of chimeras must be produced in order to increase the likelihood of achieving germline transmission. A succession of pregnancies must be completed before the correct sperm or ova come through in the germline. Using methods of the invention, efficient germline transmission can be achieved using any ES cell line.
As transgenic gametes, i.e. sperm or ova, are selected for use in fertilisation rather than the process being left to chance, then germline transmission is achieved with high efficiency, regardless of the cell line used or quality of chimera. Thus, using methods of the invention efficient germline transmission can be achieved from both poor ES cells and low percentage chimeras.
Examples
A DNA construct is made containing a ubiquitous promoter coupled to a fluorescent protein, enabling this protein to be expressed in all tissues, but most importantly in the germ cells.
Conventionally this is achieved by pronuclear injection. However a lentiviral approach could be employed (Lois et ai (supra)). The fluorescent mice produced are mated to obtain blastocysts (Day 3.5) which are removed for subsequent injection.
Targeted embryonic stem cells from a different strain of mouse or the same strain, into which foreign DNA has been introduced, are injected into blastocysts. The injected blastocysts are implanted into a recipient female mouse and develop into chimeric progeny*
A chimeric progeny pup has two types of germ cells, derived either from the fluorescent protein donor mouse blastocyst or from the targeted embryonic stem cells. Sperm is collected from a male chimera, the quality of the chimera can be of a very low percentage. The sperm of the chimera are sorted by FACS and those sperm not fluorescing, i.e. those with the transgene rather than the fluorescent marker, are used to generate germline transmission by either IVF or ICSI.
The methods should be carried out by competent personnel, proficient in mouse embryology to such a standard as to be proficient in embryo handling, ES cell injection and IVF in the mouse. FACS sorting can be carried out by different personnel and this facility is available commercially.
Transgenic blastocysts are used to create knockout mice via ES cell injection. The strain of mice from which the blastocysts are derived, C57BL 6 TG (ACTBEGFP) 1 OSB/J, stock# 003291 (JAX Mouse Lab, USA), has a ubiquitous green fluorescent protein (GFP) expressed in ail cells. Green fluorescent blastocysts are obtained and injected with non fluorescing ES cells to create a mosaic mouse (chimera) in which the blastocyst derived sperm cells express green fluorescent protein and the genetically modified sperm, derived from the ES cell, do not produce green fluorescent protein.
GFP mouse day 4 embryos (blastocysts) are produced by mating two GFP mice together and looking for the presence of a vaginal plug to indicate that mating has taken place. Female mice are sacrificed on the morning of day 4 (day 1 = day of plug). The uterus is dissected and placed in saline or FHM medium (Methods in Enzymology 225:153-164) at room temperature. Embryos are flushed out of the uterus with FHM media and collected in a Petri dish. They are then harvested from the Petri dish using small glass pipettes and placed into drops of FHM under paraffin oil (Sigma: M8410). The blastocysts are then placed in an incubator at 37°C or left on the bench at room temperature for 1 hour until use.
ES cells are transfected with foreign DNA of known sequence having a marker for selection and designed to alter a target gene of interest After homologous recombination, correctly targeted clones are expanded for injection into the GFP blastocysts.
GFP blastocysts with large blastocele cavities are selected and the transformed . ES cells are injected into the cavity. This is carried out using a micro- manipulation system that allows precise manipulation to be carried out, resulting in an embryo which is a GFP blastocyst with non-GFP ES cells within its blastocel. Typically 6-20 cells are placed in each blastocyst. These embryos are then placed (in a group of 10-12 embryos) into the uterus of a pseudopregnant recipient mouse. Pseudopregnancy is induced by allowing a female mouse to mate with an infertile (vasectomised) male mouse. The act of copulation (signified by the presence of a vaginal plug) at the time of oestrus allows the uterus to be primed to accept an embryo. To achieve pregnancy, the embryo is implanted in the uterus, either the day after plug formation (day 1) or on day 3 following plug formation, using the following procedure.
The recipient mouse is anaesthetised and the uterus withdrawn from the body. The embryos are placed into the uterus via a small glass needle through a small puncture hole in the side of the uterus. The embryos are drawn up the lumen of the needle and placed into the lumen of the uterus. The needle is withdrawn and the uterus replaced into the body cavity. The skin is closed with suture clips and the mouse is allowed to recover.
After a gestation of 3 weeks the pups are delivered. Chimeric mice can be identified as they are black/agouti mosaics, because the GFP mouse strain from which the blastocyst ceils are derived has black fur and the mouse strain from which the ES cells are derived have agouti fur. Male chimeras produce two populations of sperm: one population being derived from the black (GFP) mouse cells and the other derived from the ES cell having the genetic modification of interest.
Traditionally, the chimeric mouse would be paired to a number of females and the pups analysed, e.g. by PCR or Southern blotting, to see if transgenic offspring have been made; confirmation that a mouse had failed to produce transgenic sperm could be obtained only after exhaustive pairings. However, in this method, sperm from male chimera is collected and examined. Chimeric sperm is recovered from the chimera by culling the male chimeric animal and removing one or both vas deferens. Alternatively, the mouse can be anaesthetised and an operation performed to remove one vas deferens. Sperm is squeezed from the vas deferens and allowed to swim into a small amount of HTF fertilization media (Quinn. (1985) Fertility and Sterility, 44; 493-498).
Sperm that do not carry the desired genetic modification are derived from the GFP mouse cell and will fluoresce green after exposure to light at an appropriate excitation wavelength. Sperm carrying the desired genetic modification (derived from the ES cell) is not fluorescent on exposure to light at the excitation wavelength.
Hand sorting or FACS sorting is used to separate the fluorescent and non- fluorescent sperm to isolate the non-fluorescent sperm having the desired genetic modification.
At the same time eggs are recovered from a female mouse which has been superovulated. Superovulation is achieved by administering two hormone injections: Pregnant Mares Serum (PMS) at 5 international units followed 48 hours later by Human Chorionic Gonadotropin (HCG) at 5 international units, to prime the young female (4 weeks old) mouse to ovulate producing a great many oocyteε, typically 40-80 depending on the mouse strain used. The egg masses are easily removed from the oviducts and are mixed with the isolated non- fluorescent sperm in vitro (IVF using HTF medium) to produce mouse embryos. The in vitro fertilisation process is performed over a 3 hour period in HTF medium in an incubator at 37°C using a 5% CO2 atmosphere. The incubated embryos are eventually implanted, at 2 cell or blastocyst stage, into a pseudopregnant recipient donor mother (as described above) and the genetically modified embryos are grown to birth. The transgenic mice produced are the founders of a unique line of mice.

Claims

1. A method for selecting a genetically modified gamete obtained from a chimeric non-human animal producing heterogeneous gametes, characterised by sorting gametes having a first genetic modification enabling expression of a first selectable marker from gametes that do not express the first selectable marker.
2. A method according to claim 1, wherein the heterogeneous gametes have a first genetic modification to enable expression of a first selectable marker or have no genetic modification.
3. A method according to claim 1, wherein the heterogeneous gametes have a first genetic modification to enable expression of a first selectable marker or have a second genetic modification.
4. A method according to claim 3, wherein the second genetic modification enables expression of a second selectable marker; and gametes are sorted by means of a or the selectable marker(s).
5. A method for selecting a genetically modified non-human animal gamete comprising: (a) providing a chimeric non-human animal producing heterogeneous gametes, said gametes: (i) having a first genetic modification enabling expression of a first selectable marker, or, (ii) not expressing the first selectable marker; (b) collecting gametes from said chimeric non-human animal, and, (c) sorting gametes having a first genetic modification enabling expression of a first selectable marker from gametes that do not express the first selectable marker.
6. A method according to claim 5, wherein the heterogeneous gametes have a first genetic modification to enable expression of a first selectable marker or have no genetic modification.
7. A method according to claim 5, wherein the heterogeneous gametes have a first genetic modification to enable expression of a first selectable marker or have a second genetic modification.
8. A method according to claim 7, wherein the second genetic modification enables expression of a second selectable marker; and gametes are sorted by means of a or the selectable marker(s).
9. A method for producing a ' genetically modified non-human animal comprising selecting a genetically modified non-human gamete using a method according to any one of claims 1 to 8 and using said gamete to generate a genetically modified non-human animal.
10. A method according to claim 9, wherein said genetically modified non- human animal is generated by IVF or ICSI.
11. A method for producing a genetically modified non-human animal comprising: (a) providing an early embryo having a first genetic modification to enable expression of a first selectable marker, (b) introducing into said early embryo a pluripotent cell: (i) having no genetic modification; or, (ii) having a second genetic modification; or, (iii) having a second genetic modification to enable expression of a second selectable marker; (c) implanting the early embryo obtained in (b) into a female host to obtain chimeric progeny, (d) collecting gametes from chimeric progeny obtained in (c), (e) sorting gametes by means of a or the selectable marker(s), and, (f) using selected gametes to generate a genetically modified non-human animal.
12. A method for producing a genetically modified non-human animal according to claim 11 comprising: (a) providing an early embryo having a first genetic modification to enable expression of a first selectable marker, (b) introducing into said early embryo a pluripotent cell having a second genetic modification, (c) implanting the early embryo obtained in (b) into a female host to obtain chimeric progeny, (d) collecting gametes from chimeric progeny obtained in (c), (e) sorting gametes by means of the first selectable marker, and, (f) using a gamete having the second genetic modification to generate a genetically modified non-human animal.
13. A method any one of the preceding claims, wherein the gamete is a sperm.
14. A method according to any one of claims 1 to 12, wherein the gamete is an ovum.
15. A method according to any preceding claim, wherein the first genetic modification is an insertion of exogenous DNA comprising a selectable marker or a substitution of endogenous DNA with exogenous DNA comprising a selectable marker.
16. A method according to claim 15, wherein the exogenous DNA further comprises one or more exogenous gene(s).
17. A method according to claim 16, wherein the one or more exogenous gene(s) is operabiy linked to the selectable marker.
18. A method according to any one of claims 3, 7 or a claim dependent thereon, wherein the second genetic modification is an insertion of exogenous
DNA, a substitution of endogenous DNA with exogenous DNA, or a deletion of endogenous DNA.
19. A method according to claim 4, 8, or a claim dependent thereon, or claim 18, wherein the second genetic modification fs an insertion of exogenous DNA comprising a second selectable marker or a substitution of endogenous DNA with exogenous DNA comprising a second selectable marker.
20. A method according to claim 19, wherein the exogenous DNA further comprises one or more exogenous gene, preferably operabiy linked to said second selectable marker.
21. A method according to claim 20, wherein said one or more exogenous gene is operabiy linked to said second selectable marker.
22. A method according to any one of claims 11 to 21 , wherein the pluripotent cell is a stem cell.
23. A method according to claim 22, wherein the pluripotent cell is an embryonic stem cell.
24. A method according to any one of claims 11 to 23, wherein the early embryo is a blastocyst.
25. A method for producing a genetically modified non-human animal comprising: (a) providing a blastocyst having a first genetic modification to enable of expression of a selectable marker, (b) introducing into said blastocyst a ES cell having a second genetic modification into said blastocyst, (c) implanting the ES-blastocyst into a female host and generating chimeric progeny, (d) collecting sperm from male chimeric progeny, (e) sorting sperm by means of the selectable marker into sperm expressing the first selectable marker and sperm having the second genetic modification, and, (f) using sperm having the second genetic modification to generate genetically modified progeny, preferably by IVF or ICSI.
26. A method according to any preceding claim, wherein the first and/or second selectable marker is a fluorescent marker or is labelled with a fluorescent moiety.
27. A method according to claim 26, wherein the first and/or second fluorescent marker is a fluorescent protein.
28. A method according to claim 27, wherein the or a fluorescent protein is selected from the group comprising a red, orange, yellow, yellow-green, green- yellow, green or cyan fluorescent protein.
29. A method according to claim 28, wherein the first or second fluorescent protein is a green fluorescent protein.
30. A method according to any one of claims 27 to 29, wherein the first and/or second fluorescent protein is a wild type, enhanced, destabilised enhanced, or red-shift fluorescent protein.
31. A method according to claim 26, wherein the first and/or second selectable marker is a surface antigen labelled directly or indirectly with a fluorescent moiety.
32. A method according to claim 31, wherein the fluorescent moiety is a fluorescent labelled molecule, preferably a fluorescent labelled antibody or a fluorescent labelled antigen binding fragment of an antibody, or a fluorescent labelled antigen binding fragment of a protein.
33. A method according to any one of claims 26 to 32, wherein the gametes are sorted into fluorescent and non-fluorescent samples, or into samples fluorescing at different wavelengths, by hand or by an automated method.
34. A method according to claim 33, wherein the automated method is FACS analysis.
35. A method according to any preceding claim, wherein the genetically modified non human animal is a horse, cow, primate, sheep, goat, pig, dog, cat, chicken, rabbit, fish, or a rodent.
36. A method according to claim 35, wherein the genetically modified non human animal is a rat or mouse.
37. A method for producing a genetically modified rodent comprising: (a) providing a blastocyst having a first genetic modification to enable expression of a selectable marker, (b) introducing into said blastocyst an ES cell having a second genetic modification, (c) implanting the ES-blastocyst into a female host and generating a chimeric male progeny rodent, (d) collecting sperm from said male chimeric progeny rodent, (e) sorting sperm by means of the selectable marker into sperm expressing the selectable marker and sperm having the second genetic modification, and (f) using sperm having the second genetic modification to generate a genetically modified progeny rodent
38. A method according to claim 37, wherein the selectable marker is a fluorescent protein, preferably a green fluorescent protein.
39. A method for producing a geneticaily modified mouse comprising: (a) providing a mouse blastocyst having a first genetic modification to enable expression of a green fluorescent protein, (b) introducing into said blastocyst a mouse ES cell having a second genetic modification, (c) implanting the ES-blastocyst into a female mouse and generating a chimeric male progeny mouse, (d) collecting sperm from said mate chimeric progeny mouse, (e) sorting sperm by means of the selectable marker into sperm expressing the selectable marker and sperm having the second genetic modification, and, (f) using sperm having the second genetic modification to generate genetically modified progeny.
40. A genetically modified gamete obtainable or obtained using a method according to any one of claims 1 to 8.
41. A genetically modified non-human animal obtained according to a method of any one of claims 9 to 39.
42. The use of a genetically modified gamete according to claim 40 in the production of a genetically modified non-human animal.
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WO2020102565A3 (en) * 2018-11-14 2020-07-30 Flagship Pioneering Innovations V, Inc. Systems and methods for nondestructive testing of gametes

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