A method of producing a conditional knockout animal, and techniques related thereto, e.g., a method of efficiently producing a floxed animal, are provided. By introducing recombinase recognition sequences such as loxP into both ends of a target region on a chromosome at different timings, an animal having the pair of recombinase recognition sequences on the chromosome, such as a floxed animal, is produced.

The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

Compositions and methods comprising bioenergetic agents for restoring the quality of aged oocytes, enhancing oogonial stem cells or improving derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in fertility-enhancing procedures, are described.

Compositions and methods comprising bioenergetic agents for restoring the quality of aged oocytes, enhancing oogonial stem cells or improving derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in fertility-enhancing procedures, are described.

An object of the present invention is to provide a method of conveniently producing a genetically modified non-human mammal with high efficiency using a CRISPR-Cas9 system and particularly a production method whereby gene knock-in can be achieved with high efficiency regardless of the gene size. The method of producing a genetically modified non-human mammal comprises introducing a Cas9 protein, a crRNA fragment comprising a nucleotide sequence complementary to a target DNA region, and a tracrRNA fragment into a non-human mammalian oocyte to genetically modify the target DNA.

A method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing the aggregate to obtain an artificial blastocyst having a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. Also, a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a foetus or a live animal and an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

A method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing the aggregate to obtain an artificial blastocyst having a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. Also, a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a foetus or a live animal and an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

The present disclosure relates to methods for making genetic edits in vitro in a non-human vertebrate cell or embryo at a plurality of target chromosomal DNA sites. Methods for making a non-human animal having multiplex genetic edits at a plurality of target chromosomal DNA sites and making a non-human vertebrate animal chimeric for host cells and donor cells are also considered.

The present disclosure relates to methods for making genetic edits in vitro in a non-human vertebrate cell or embryo at a plurality of target chromosomal DNA sites. Methods for making a non-human animal having multiplex genetic edits at a plurality of target chromosomal DNA sites and making a non-human vertebrate animal chimeric for host cells and donor cells are also considered.