Patent Application
20220186261
N/A
Animal selection has traditionally been performed using phenotypic records and pedigrees in which superior animals are chosen as parents according to an estimated breeding value (EBV). Such traditional strategies have been successful for traits with high heritability, e.g. genetic improvement for milk yield has increased consistently for many decades. However, progeny testing and pedigree information has proven less effective with traits with low heritability. Moreover, an accurate EBV is costly and time consuming to obtain due to the recording and analysis of many individuals and to the long generation intervals in cattle. Indeed, identifying elite dairy sires by EBV relies on a tedious progeny-testing scheme that takes 6-7 years and costs approximately $50,000 per bull tested.
Recent advances in molecular genetics have enabled a novel selection strategy for identifying genetically superior parents by the use of DNA markers associated with quantitative traits (Meuwissen et al. 2001). A key breakthrough in marker-assisted selection came with the sequencing of the whole bovine genome (Bovine HapMap et al. 2009), which has led to the discovery of many thousands of DNA markers in the form of single nucleotide polymorphisms (SNP) associated with production traits and other traits of interest. These novel molecular tools have enabled a dramatic reduction in the cost of genotyping.
A second major breakthrough came with the demonstration that it is possible to make accurate selection decisions when breeding values are predicted from DNA markers alone by calculating genomic breeding values (GEBV).
The implications of achieving accurate GEBV for animals at birth are profound. Potentially, genomic selection can lead to a doubling of the rate of genetic gain through selection and breeding from bulls at 2 years of age rather than 5 years of age or later (Schaeffer 2006). Although more genotyping is needed to increase selection intensity and thereby increase the rates of genetic gain, it is expected that cattle breeding companies can save a large majority of their costs using GEBV instead of the traditional EBV (Hayes et al. 2009). It has now become possible to evaluate the genetic merit of a newborn calf or even a preimplantation stage embryo, provided that a reference population is available, with comparable accuracy for less than $100 (Georges 2014).
Although cattle generation intervals can be dramatically decreased and selection accuracy can be greatly improved by using the genomic approach and selecting genetically superior offspring very early post-fertilization, selection programs are consistently limited by independent assortment and crossing over of parental chromosomes during meiosis, causing uncontrollable genomic variability prior to fertilization.
Meiotic genetic diversity is ensured during two events, crossing over and independent assortment of chromosomes. Crossing over occurs during prophase I of meiosis, and enables homologous pairs of chromosomes to recombine and often exchange chromosome segments. This allows genes from each parent to intermix and create chromosomes with a different genomic complement. Independent chromosome assortment occurs during meiosis II when sister chromatids separate and are randomly distributed to the daughter cells, i.e. gametes. In cattle, independent assortment can yield 230, or 1,073,741,824, unique ways to arrange 30 pairs of chromosomes. To date, all selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes prior to fertilization. Therefore, we propose the present invention, wherein haploid cell lines derived preferably from male and female gametes, or somatic cells, can be obtained and analyzed to select those carrying superior genomic markers prior to fertilization. The most promising haploid cells can then be ‘reconstructed,’ i.e. fertilized, to produce embryos and offspring with predetermined genomes.
Diploid genomes (two sets of chromosomes, one maternal and one paternal) are typical among most living animals, and haploidy (a single set of chromosomes) is generally limited to gametes. Although a diploid genome is thought to increase fitness by masking mutations, it also leads to the accumulation of mutations with time. To counteract long-term degradation of the genome, mammals created adaptations that include genomic imprinting, random monoallelic expression and X chromosome inactivation (Wutz 2014). Haploidy is normally restricted to the post-meiotic stages of germ cells and represents the end point of cell proliferation, which means that physiological haploidy is incompatible with self-renewal.
However, the recent advent of haploid mouse embryonic stem cell (ESC) technologies has drastically changed this situation (Kokubu & Takeda 2014). Recently, some studies have looked at deriving ESCs from mammalian parthenogenetic and androgenetic haploid embryos. These initial studies were focused on mouse ESCs (Elling et al. 2011; Leeb & Wutz 2011; Li et al. 2012; Yang et al. 2012). Similar techniques have been applied to monkey (Yang et al. 2013) and rat haploid ESC derivation (Li et al. 2014).
The original versions of haploid ESC lines (Elling et al. 2011; Leeb et al. 2014) were generated by parthenogenetic activation of unfertilized mouse oocytes with chemicals such as strontium salt or ethanol. These haploid mouse ESCs contain only the maternal set of chromosomes, and show pluripotency as well as self-renewal capabilities. Androgenetic haploid mouse ESC lines containing only the paternal chromosomes have also been generated by removal of the maternal pronucleus from zygotes and by introduction of sperm into enucleated oocytes (Li et al. 2012; Yang et al. 2012). Thus, pluripotency, self-renewal, and haploidy can be incorporated together in a single cell line.
Haploid ESC lines have been shown to function as gametes and support further embryonic development (Li et al. 2012; Yang et al. 2012; Wan et al. 2013; Shuai & Zhou 2014). Metaphase oocytes were ‘fertilized’ with haploid ESC by intracytoplasmic cell injection resulting in the production of fertile pups.
In another experiment sperm were injected into an enucleated oocyte, followed by the activation of the reconstructed embryos by chemical stimulus. Pups were generated, albeit at low efficiencies (Wan et al. 2013).
Although haploid ESC have never been reported in domestic species, diploid embryonic stem-like cells have been isolated previously from in vitro fertilized, nuclear transfer and parthenogenetic diploid embryos (Stice et al. 1996; Betts et al. 2001; Talbot et al. 2005; Wang et al. 2005; Pashaiasl et al. 2010; Jin et al. 2012, Bogliotti et al, 2018). One of the major barriers in deriving genuine ESC lines in ungulate species relates to their tendency of undergoing differentiation to ‘cobblestone-like’ epithelial cells that show either lengthy cell cycles or complete cell cycle arrest after prolonged culture periods (Talbot et al. 2005; Desmarais et al. 2011). Nonetheless, recent studies have indicated that stable primed pluripotent embryonic stem cells can be obtained from bovine IVF blastocyst stage embryos (Bogliotti et al, 2018).
Such tendency to differentiate is further exacerbated when cultures are performed in the absence of feeder-layers. To eliminate the risk cell contaminants from different species, e.g. mitomycin-treated mouse embryonic fibroblasts, different types of extracellular matrix (ECM) such as lamin, gelation or combination of both, have been successfully used to derive bovine ESC lines (Verma et al. 2013). Together, the background research described above provides a base and background for the present invention.
To summarize, the present invention is aimed at reducing meiotic uncertainty from breeding programs, preferably ungulate breeding programs, by determining the genomic value of paternal and/or maternal gametes and screening or selection of those gametes before creation of a diploid embryo.
In one embodiment, the present invention is a method of generating mammalian diploid embryos and/or offspring with a pre-characterized genome comprising the steps of (a) obtaining an embryonic haploid cell, (b) deriving haploid outgrowth from the cell of step a, (c) characterizing the genome of the haploid cells of step a or b, and (d) deriving diploid embryos and/or offspring from the cells of steps a, b or c. In a preferred embodiment, the embryonic haploid cell is an ungulate cell. In another preferred embodiment, the cell is a bovine cell.
In one embodiment, the embryonic haploid cell is derived from an ungulate parthenogenetic haploid embryo in vitro cultured. The embryo may be prepared by a method comprising haploid parthenogenetic activation of an ungulate oocyte.
In another embodiment, the haploid cell is derived from an ungulate androgenetic haploid embryo in vitro cultured. The embryo may be prepared by a method comprising removing the ungulate oocyte's genome either before or after fertilization by the intracytoplasmic sperm injection (ICSI) of a single sperm or in vitro fertilization (IVF).
In one embodiment, the haploid outgrowth comprises cellular multiplication to produce a larger number of haploid cells, the method comprising the in vitro culturing of a haploid cell isolated from a preimplantation embryo. The haploid embryonic cell may isolated from a preimplantation embryo at a cleavage, four cells, eight cells, or sixteen cells, or from a morula or blastocyst stage embryo.
In one embodiment, characterizing the genome of the haploid cells comprises performing haploid genomic scoring of the haploid cell. That scoring may include (i) scoring the genomic screening the haploid embryonic-derived cell or (ii) selecting the haploid embryonic-derived cells for the preferred haploid genomic score or selection index. The score may comprise including a weighted combination of one or more single nucleotide polymorphisms. The score may be based on traits selected from the group consisting of production traits (e.g. milk, fat, protein, fat %, protein %, composition of milk protein variants), health traits (e.g. somatic cell score, mastitis resistance, immune response, livability, disease resistance), reproductive traits (e.g. pregnancy rate, conception rate), calving traits (e.g. calving ease, calving to first insemination, stillbirths), conformation traits (e.g. polled trait, udder and teat traits, feet and leg traits, body traits, dimension traits), efficiency traits (e.g. feed efficiency traits, workability, longevity, productive life), novel traits (e.g. robotic milking traits, heat tolerance, activity traits and behavior traits), and composite index traits (e.g. LPI (Life Production Index), and TPI (Total Production Index).
In one embodiment, obtaining the derived diploid embryos and/or offspring with a precharacterized genome may comprise introducing the genomically characterized haploid cell isolated from a haploid preimplantation embryo or outgrowth line into an oocyte.
In another embodiment, the haploid embryonic-derived cell carrying the preferred genomic score is used for maternal embryo reconstruction, the method additionally comprising introducing an ungulate haploid embryonic-derived cell isolated from a parthenogenetic haploid preimplantation embryo or outgrowth line into a fertilized and enucleated ungulate oocyte before paternal pronuclear formation.
In one embodiment, the haploid embryonic-derived cell carrying the preferred genomic score is used for paternal embryo reconstruction, the method additionally comprising introducing a haploid embryonic-derived cell isolated from an androgenetic haploid preimplantation embryo or outgrowth line into a parthenogenetically-activated oocyte before maternal pronuclear formation.
In another embodiment, the haploid embryonic-derived cells carrying the preferred genomic score are used for biparental embryo reconstruction, the method additionally comprising introducing both a haploid embryonic-derived cell isolated from an androgenetic haploid preimplantation embryo or outgrowth line and an ungulate haploid embryonic-derived cell isolated from a parthenogenetic haploid preimplantation embryo or outgrowth line into an oocyte.
In one embodiment, offspring are derived by a method comprising implanting the reconstructed diploid into a recipient host. In another embodiment, the introducing of the haploid cell into the oocyte comprises electrofusion or cell injection using micromanipulation approaches. In preferred embodiments, the haploid embryonic-derived cell is isolated from a preimplantation embryo at the cleavage, four cells, eight cells, sixteen cells, morula, or blastocyst stages, or isolated from a haploid outgrowth.
In one embodiment, the cells of step a, b, or c are characterized by screening for preferred genetic or genomic characteristics. The screening may be selected from the method selected from the group consisting of:
In one embodiment, the screening or selection of the cells of step (a), (b), or (c) are selected after assessment of homozygosity of the cells and/or their genomic imputation and estimation genomic breeding value of each haploid embryo.
In one embodiment, screening or selection is done to:
In one embodiment, screening or selection is done to create breeding animals or breeding lines that contain a unique combination of alleles, haplotypes, or traits, wherein the alleles, haplotypes, or traits are normally appearing at low frequency in the population, wherein the frequency is normally below 50% of the population.
In one embodiment, the genetic or genomic characteristics are selected from the group consisting of production traits (e.g. milk, fat, protein, fat %, protein %, milk protein variant composition), health traits (e.g. somatic cell score, mastitis resistance, immune response, livability, disease resistance), reproductive traits (e.g. pregnancy rate, conception rate), calving traits (e.g. calving ease, calving to first insemination, stillbirths), conformation traits (e.g. polled traits, udder and teat traits, feet and leg traits, body traits, dimension traits), efficiency traits (e.g. feed efficiency traits, workability, longevity, productive life), novel traits (e.g. robotic milking traits, heat tolerance, activity traits and behavior traits), and composite index traits (e.g. LPI (Life Production Index) and TPI (Total Production Index).
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, mammalian embryo culture, molecular biology, cell biology and gamete micromanipulation, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd edition; F. M. Ausubel, et al. eds. (1987) Current Protocols In Molecular Biology; the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.); and R. I. Freshney, ed. (1987) Animal Cell Culture.
All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (−) by increments of 1.0 or 0.1, as appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.
As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
As used herein, “ploidy” is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and sexual genes. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present (the “ploidy level”): haploid (1 set), diploid (2 sets), triploid (3 sets), tetraploid (4 sets), pentaploid (5 sets), etc. The generic term “polyploid” is often used to describe cells with three or more chromosome sets.
“Diploid” is meant to indicate a cell, e.g., a gamete or blastomere, having two sets of chromosomes of paternal, maternal or both origins.
“Haploid” is meant to indicate a cell, e.g., a gamete or blastomere having one set of chromosomes of paternal or maternal origin.
“Oocyte activation” indicates wherein a fertilized or unfertilized oocyte, preferably in metaphase II of meiosis, undergoes a process typically including extrusion of the second polar body, exocytosis of cortical granules (CGs), meiotic cell-cycle resumption, pronuclear formation, translation of maternal mRNAs, and meiosis-to-mitosis transition. In the present invention, “oocyte activation” refers to methods whereby an ungulate oocyte containing DNA of paternal and/or maternal origin is induced to develop by natural or artificial fertilization and/or through mechanical, chemical, and/or electrical stimuli into an embryo that has a discernible inner cell mass and trophectoderm. Methods of performing oocyte activation are known in the art. See, for example, Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(Suppl. 1)11911-6.
“Metaphase II” stage of cell cycle is wherein the DNA content of an oocyte consists of a haploid number of chromosomes with each chromosome represented by two chromatids.
“Anaphase II”/“Telophase II” refers to the transition-phase of the meiosis II characterized by an asymmetrical cell division, bilateral furrowing, and abscission of the polar body, where the chromatids of each chromosome move to opposite poles still joined on the spindle.
“ICSI,” or “intracytoplasmic sperm injection,” refers to the process wherein one or more sperm are injected directly into an egg using micromanipulation approaches.
By “IVF” or “in vitro fertilization” is meant the process of fertilization wherein an oocyte is combined with sperm outside the body, in vitro.
“Enucleation” refers to removing the ungulate oocyte's genome as aided by micromanipulation techniques.
A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular genotype, it is generally preferable to use a positive control (e.g., a sample from a subject, carrying such alteration and exhibiting the desired phenotype), and a negative control (e.g., a subject or a sample from a subject lacking the altered expression or phenotype).
As used herein, the terms “culture media” and “culture medium” are used interchangeably and refer to a liquid substance used to support the growth of cells (e.g., mammalian embryonic cells). Preferably, the culture media as used herein can be a water-based media including a combination of ingredients such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors, and hormones. For example, a culture media can be a synthetic culture media such as, for example, synthetic oviductal fluid media (SOF), modified SOF, KSOM media (MilliporeSigma, Burlington, Mass., USA), modified KSOM, minimum essential media (MEM) (HyClone Thermo Scientific, Waltham, Mass., USA), DMEM/F12 (Life Technologies, Carlsbad, Calif., USA), Neurobasal Medium (Life Technologies, Carlsbad, Calif., USA), KO-DMEM (Life Technologies, Carlsbad, Calif., USA), DMEM/F12 (Life Technologies, Carlsbad, Calif., USA), supplemented with the necessary additives as is further described herein. In some embodiments, the cell culture media can be a mixture of culture media. Preferably, all ingredients included in the culture media of the present disclosure are substantially pure and tissue culture grade.
The term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.
The term “diploid embryo reconstruction” refers to establishing a diploid embryo from a maternal diploid reconstruction process wherein the maternal diploid reconstruction process comprises (a) inserting a mammalian, preferably an ungulate, haploid parthenogenetic cell into a fertilized and enucleated oocyte, (b) establishing a diploid embryo from a paternal diploid reconstruction process, wherein the paternal diploid reconstruction process comprises inserting the haploid androgenetic embryonic cell into a parthenogenetically-activated oocyte, and (c) establishing a diploid embryo from a biparental diploid reconstruction process, wherein the biparental diploid reconstruction process comprises inserting a haploid parthenogenetic and an androgenetic embryonic cell into an enucleated and activated oocyte.
An ungulate is a hoofed animal, e.g., a bovine, an ovine, an equine, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros.
The present disclosure is a method of using haploid embryonic cells to characterize haploid genotypes. In a preferred embodiment, the method is used to generate offspring with predetermined genomes or genetic characteristics.
In one embodiment, the method of the present invention comprises four phases, as described below. In brief, one will obtain haploid embryonic cells (either androgenetic or parthenogenetic cells) in phase 1, derive haploid embryonic outgrowth (
One preferred goal of the present invention is to obtain preferred genetic characteristics in an embryo or offspring. For example, one may wish to obtain the following characteristics in a bovine embryo: in some embodiments of the invention, we would look at production traits (fat, protein, milk production, milk protein variant composition, e.g. A2A2 milk), health traits (e.g. somatic cell score, mastitis resistance, metabolic disease resistance, reproductive disease resistance, immune response, livability or reduced incidence of various diseases), reproductive traits (e.g. daughter pregnancy rate, sire conception rate), calving traits (e.g. calving ease, calving to first insemination, stillbirths), conformation traits (e.g. udder and teat traits, feet and leg traits, body traits, dimension traits), efficiency traits (e.g. feed efficiency traits, workability, longevity, productive life), novel traits (e.g. polled trait, heat tolerance, robotic milking traits, activity traits and behavior traits), LPI (Life Production Index—more popular in Canada), or TPI (Total Production Index—more popular in USA) Productive Life. In another version of the present invention, one may wish to eliminate undesired traits (e.g. horned trait, various deleterious haplotypes, etc.).
The method of the present invention is useful for any mammalian species (preferably non-human mammals) or, more preferably, any ungulate species. A preferred version of the invention uses commercially important species such as bovine, equine, ovine, caprine, and porcine species. Most preferably, one would use bovine gametes (sperm and oocytes) for the phases. One could also use somatic cell lines that are de-differentiated into pluripotent cells and use the approach described herein to produce haploid cells.
The examples below disclose exemplary or preferred methods of achieving the phases. Of course, other methods may be preferable in some circumstances. For example, the present invention includes the use of fully mature gametes, both male (sperm) or/and female (oocytes).
Another embodiment would include the use of other post-meiotic male (i.e. spermatocytes and spermatids) and female (i.e. second polar bodies) gametes/cells to produce the haploid cells or cell lines. One could also use pre-meiotic cells (i.e. spermatogonia, oogonia or stem cells) and then enable meiotic resumption in vitro to produce the haploid cells or cell lines. Additionally (and as mentioned previously), one could induce meiosis in any somatic cell (stem cell or not).
Phase 1—Derivation of Haploid Embryonic Cells.
Rationale: Multiple haploid embryonic cells are obtained from paternal (sperm) and maternal (oocyte) individual gametes by androgenetic and parthenogenetic development, respectively.
Exemplary Methodology:
Note: the experimental protocol below is based on our actual experimental work with bovine haploid androgenetic (Phase 1a) and parthenogenetic (Phase 1b) embryonic cells, derivation of haploid embryonic outgrowths (Phase 2), genomic screening and selection (Phase 3), and derivation of diploid embryos with characterized parental genomes using haploid embryonic cells (Phase 4). The description of the derivation of offspring below is prophetic. We have offered substitutions and suggestions for variant method steps based on our vision of prophetic embodiments.
Phase 1a—Derivation of Haploid Androgenetic (Sperm-Derived) Embryonic Cells (
Step 1. Preparation of in vitro matured (MII) bovine oocytes (similar to Hansen 2013):
Step 2. Intracytoplasmic bovine sperm injection (ICSI) of oocytes (similar to Matcuit et al, 2006)
Step 3. Removal of the chromosomes from ICSI-derived zygotes (oocyte enucleation as described by Bordignon, Smith, 1998). Note that we could also remove the chromosomes from IVF-derived (conventional in vitro fertilization) fertilized zygotes.
Step 4. Culture of the androgenetic haploid zygote (similar to Hansen 2013):
Phase 1b—Derivation of Haploid Parthenogenetic (Oocyte-Derived) Embryonic Cells.
Methodology (
Step 1. Preparation of in vitro matured (MII) oocytes (as described in Phase 1a):
Step 2. Haploid parthenogenetic activation (similar to Alberio et al, 2001 and Suvá et al, 2019):
Step 3. Culture of the parthenogenetic haploid zygote (as above)
Results:
The results described below demonstrate that ungulate (specifically bovine) haploid embryos containing multiple embryonic cells can be obtained by the in vitro culture of both androgenetic and parthenogenetic zygotes (Table 1 and
Phase 2—Derivation of Haploid Embryonic Outgrowth and Cell Lines (
Rationale: Haploid embryonic cell lines derived from multicellular ungulate androgenetic and parthenogenetic embryos can be cultured in vitro for prolonged periods to derive an even larger number of haploid embryonic cells.
Methodology:
Results:
Results in Table 2 and
Phase 3—Genomic Characterization of Haploid Androgenetic and Parthenogenetic Embryonic Cells.
Rationale: Genomic characterization can be obtained from a small sample of androgenetic and parthenogenetic haploid embryonic cells. In a preferable method of the present invention, the target cells are characterized by screening, scoring, or selecting for preferred genetic or genomic characteristic. “Screening” typically refers to detecting (preferably through genotyping) the presence or absence of certain commercially valuable traits. “Scoring” typically refers to screening for multiple traits at once, then creating a score that is made up of a series of traits that are equally or differentially weighted according to their relative value. Selection then refers to identifying a subpopulation of animals in the population with the highest scores. “Selection” can also refer to a genomic value made up of hundreds or thousands of genomic markers that span the entire genome and constitute a genomic prediction of the breeding value or transmitting ability of an individual. A subpopulation of animals is then selected on the basis of their genomic breeding values or genomic transmitting ability.
In one embodiment, the cells are scored wherein characterizing the genome of the haploid cells comprises performing haploid genomic scoring of the haploid cell. The haploid genomic scoring may comprise (i) genomically screening the haploid embryonic-derived cell or (ii) selecting the haploid embryonic-derived cells for the preferred haploid genomic score.
In another embodiment, the haploid genomic score comprises including a weighted combination of one or more of one or more single nucleotide polymorphisms.
In one embodiment, the cells are screened via methods selected from the group consisting of:
In another version of the method, the screening or selection of the cells is after assessment of homozygosity of the cells and/or their genomic imputation and estimation genomic breeding value of each haploid embryo.
In one version of the method, screening, scoring, or selection is done to:
In another preferred version of the invention, the genetic or genomic characteristics are selected from the group consisting of production traits (e.g. milk, fat, protein, fat %, protein %, milk protein variant composition), health traits (e.g. somatic cell score, mastitis resistance, immune response, livability, disease resistance), reproductive traits (e.g. pregnancy rate, conception rate), calving traits (e.g. calving ease, calving to first insemination, stillbirths), conformation traits (e.g. polled traits, udder and teat traits, feet and leg traits, body traits, dimension traits), efficiency traits (e.g. feed efficiency traits, workability, longevity, productive life), novel traits (e.g. robotic milking traits, heat tolerance, activity traits and behavior traits), and composite index traits (e.g. LPI (Life Production Index), and TPI (Total Production Index).
Typical Methodology:
Results:
Phase 4—Diploidization: Derivation of Ungulate Diploid Embryos and Offspring from Androgenetic and/or Parthenogenetic Embryonic Cells.
Rationale: Diploid embryos and viable offspring can be obtained from genomically characterized haploid androgenetic and/or parthenogenetic embryonic cells.
Phase 4a—Derivation of Diploid Embryos and Offspring from Androgenetic Haploid Embryonic Cells (Paternal Embryo Reconstruction).
Methodology (see
Step 1. Genomically characterized haploid androgenetic cells (Phases 1a or 2, and 3) are dissociated to obtain individualized cells;
Step 2. Haploid androgenetic cells are introduced into the cytoplasm of TII oocytes (with a second polar body) after parthenogenetic activation and before maternal pronuclear formation. Note: Haploid nuclear introduction can be performed by microinjection (as in ICSI) or by fusion of the plasma membrane of the haploid cell to the oocyte. Alternatively, one could use MII oocytes.
Step 3. After the introduction of haploid androgenetic nuclei, reconstructed diploidized zygotes carrying a genomically characterized paternal genome are cultured in vitro (as previously described above) for 7 days. Reconstructed embryos of good quality are either transferred fresh or frozen (to be transferred at a later date) using conventional embryo transfer protocols as described in George E. Seidel, Jr. and Sarah Moore Seidel's Training manual for embryo transfer in cattle, FAO Animal Production and Health, Paper 77.
Phase 4b—Derivation of Diploid Embryos and Offspring from Parthenogenetic Haploid Embryonic Cells (Maternal Embryo Reconstruction).
Methodology (see
Step 1. Genomically characterized haploid parthenogenetic cells (Phases 1b or 2, and 3) are dissociated to obtain individualized cells;
Step 2. Disaggregated individualized haploid parthenogenetic cells are introduced into the cytoplasm of an enucleated zygote at approximately 2 to 10 h after fertilization and/or sperm penetration in the oocyte (ICSI or IVF). Note: Haploid nuclear introduction can be performed by microinjection (as in ICSI) or by fusion of the plasma membrane of the haploid cell to the oocyte.
Step 3. After the introduction of parthenogenetic haploid nuclei, reconstructed diploidized zygotes carrying a genomically characterized maternal genome will be cultured in vitro (as previously described above) for 7 days;
Phase 4c—Derivation of Diploid Embryos and Offspring from Androgenetic and Parthenogenetic Haploid Embryonic Cells (Biparental Embryo Reconstruction).
Methodology (see
Step 1. Genomically characterized haploid androgenetic and parthenogenetic cells (Phases 1a-b, 2a-b, and 3) will be dissociated to obtain individualized cells, most likely by the following method:
Step 2. Disaggregated individualized haploid androgenetic and parthenogenetic cells are introduced into the cytoplasm of an oocyte enucleated at TII (with a second polar body) between 2 to 15 h after parthenogenetic activation, most likely by the following method. Note: Haploid nuclear introduction can be performed by microinjection (as in ICSI) or by fusion of the plasma membrane of the haploid cell to the oocyte.
Step 3. After the introduction of both haploid nuclei, reconstructed diploidized zygotes carrying a genomically characterized paternal and maternal genome are cultured in vitro (as previously described in sections [0030] and [0035]) for 7 days;
Results:
These results demonstrate that bovine diploid embryos produced by reconstructing of oocytes using haploid embryonic cells will cleave at a rate similar to fertilized controls and develop to the blastocyst stage in vitro (Table 3 and
One may wish to clone or multiply the reconstructed diploid embryo to make multiple copies of the embryo. For example, we have used the ECNT protocols reported in the literature, which includes blastomere separation from bovine embryos developed by Dr. Steen Willadsen (Willadesen, S. M. (1989) Cloning of sheep and cow embryos. Genome 31, 956-962) and production of nuclei from cultured inner cell mass cells from bovine embryos by Drs. Sims and First (Sims, M., & First, N. L. (1994) Production of calves by transfer of nuclei from cultured inner cell mass cells. Proc Natl Acad Sci USA, 91(13), 6143-6147).
This application claims the benefit of U.S. Provisional Patent Application No. 62/808,106, filed Feb. 20, 2019, titled “Use of Haploid Embryonic Cells to Generate Offspring with Predetermined Genomes,” which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/050210 | 2/19/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62808106 | Feb 2019 | US |
