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A proposal of a novel experimental procedure to genetically identify disease gene loci in humans.

Muto T - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

Bottom Line: This protocol produces a set of diploid pluripotent stem cell clones having maternal and paternal chromosomes in different manners to each other.The genetic loci for the disease genes are determined through the conventional processes of positional cloning.Thus, taking advantage of the strategy proposed here, if the abnormality is reproducible using patient-derived pluripotent stem cells, a single carrier of the genetic mutations would be adequate to identify the disease gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan. muto@cdb.riken.jp

ABSTRACT
Forward genetics in humans is beneficial in terms of diagnosis and treatment of genetic diseases, and discovery of gene functions. However, experimental mating is not possible among humans. In order to overcome this problem, I propose a novel experimental procedure to genetically identify human disease gene loci. To accomplish this, somatic cells from patients or their parents are reprogrammed to the pluripotent state, oogenesis is induced, the oocytes are parthenogenetically activated in the presence of cytochalasin, and embryonic stem cells are established from the parthenogenetic blastocysts. This protocol produces a set of diploid pluripotent stem cell clones having maternal and paternal chromosomes in different manners to each other. The genetic loci for the disease genes are determined through the conventional processes of positional cloning. Thus, taking advantage of the strategy proposed here, if the abnormality is reproducible using patient-derived pluripotent stem cells, a single carrier of the genetic mutations would be adequate to identify the disease gene loci.

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Related in: MedlinePlus

Variable inheritance of chromosomes and mutations in experimental human genetics. (A) Chromosomal segregation during parthenogenesis. Chromosomal segregation during meiosis and parthenogenesis is depicted. Bars and thick circles represent arms and centromeres of the chromosomes, respectively. Chromosomes originating from different parents are drawn in different colors (black and red). Chromosomes are inherited by p(MI) and p(MII) clones in different manners. (B, C) Various combinations of mutations and the onset of diseases. Schematic representation of various cases in polygenic diseases (B) and the cases in which mutations and genomic imprinting are located in same loci (C). Black bars and X in circles represent chromosomes and mutations, respectively. Red X, dominant mutation; blue X, recessive mutation; Red circle, affected patients or clones. Red triangles represent mutations that lead to 50% inactivation of the genetic loci, and black squares represent genetic loci inactivated by genomic imprinting. For explanation of each case, refer to text.
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fig03: Variable inheritance of chromosomes and mutations in experimental human genetics. (A) Chromosomal segregation during parthenogenesis. Chromosomal segregation during meiosis and parthenogenesis is depicted. Bars and thick circles represent arms and centromeres of the chromosomes, respectively. Chromosomes originating from different parents are drawn in different colors (black and red). Chromosomes are inherited by p(MI) and p(MII) clones in different manners. (B, C) Various combinations of mutations and the onset of diseases. Schematic representation of various cases in polygenic diseases (B) and the cases in which mutations and genomic imprinting are located in same loci (C). Black bars and X in circles represent chromosomes and mutations, respectively. Red X, dominant mutation; blue X, recessive mutation; Red circle, affected patients or clones. Red triangles represent mutations that lead to 50% inactivation of the genetic loci, and black squares represent genetic loci inactivated by genomic imprinting. For explanation of each case, refer to text.

Mentions: Oocytes that are arrested at the first meiotic prophase or second meiotic metaphase are collected and cultivated in vitro. Next, these are stimulated to induce parthenogenesis (Fig. 2C). If the oocytes are treated with cytochalasin when parthenogenetically activated to prevent extrusion of the polar bodies during meiosis, the parthenogenetic embryos keep diploidy and possess maternal and paternal chromosomes in various ratios40,41) (Figs. 1C, 3A). Parthenogenetic embryonic stem (pES) cell clones are collected from the resultant blastocysts of the parthenogenetic embryos42,43) and cultured separately in wells of culture plates (Fig. 2C).


A proposal of a novel experimental procedure to genetically identify disease gene loci in humans.

Muto T - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

Variable inheritance of chromosomes and mutations in experimental human genetics. (A) Chromosomal segregation during parthenogenesis. Chromosomal segregation during meiosis and parthenogenesis is depicted. Bars and thick circles represent arms and centromeres of the chromosomes, respectively. Chromosomes originating from different parents are drawn in different colors (black and red). Chromosomes are inherited by p(MI) and p(MII) clones in different manners. (B, C) Various combinations of mutations and the onset of diseases. Schematic representation of various cases in polygenic diseases (B) and the cases in which mutations and genomic imprinting are located in same loci (C). Black bars and X in circles represent chromosomes and mutations, respectively. Red X, dominant mutation; blue X, recessive mutation; Red circle, affected patients or clones. Red triangles represent mutations that lead to 50% inactivation of the genetic loci, and black squares represent genetic loci inactivated by genomic imprinting. For explanation of each case, refer to text.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066544&req=5

fig03: Variable inheritance of chromosomes and mutations in experimental human genetics. (A) Chromosomal segregation during parthenogenesis. Chromosomal segregation during meiosis and parthenogenesis is depicted. Bars and thick circles represent arms and centromeres of the chromosomes, respectively. Chromosomes originating from different parents are drawn in different colors (black and red). Chromosomes are inherited by p(MI) and p(MII) clones in different manners. (B, C) Various combinations of mutations and the onset of diseases. Schematic representation of various cases in polygenic diseases (B) and the cases in which mutations and genomic imprinting are located in same loci (C). Black bars and X in circles represent chromosomes and mutations, respectively. Red X, dominant mutation; blue X, recessive mutation; Red circle, affected patients or clones. Red triangles represent mutations that lead to 50% inactivation of the genetic loci, and black squares represent genetic loci inactivated by genomic imprinting. For explanation of each case, refer to text.
Mentions: Oocytes that are arrested at the first meiotic prophase or second meiotic metaphase are collected and cultivated in vitro. Next, these are stimulated to induce parthenogenesis (Fig. 2C). If the oocytes are treated with cytochalasin when parthenogenetically activated to prevent extrusion of the polar bodies during meiosis, the parthenogenetic embryos keep diploidy and possess maternal and paternal chromosomes in various ratios40,41) (Figs. 1C, 3A). Parthenogenetic embryonic stem (pES) cell clones are collected from the resultant blastocysts of the parthenogenetic embryos42,43) and cultured separately in wells of culture plates (Fig. 2C).

Bottom Line: This protocol produces a set of diploid pluripotent stem cell clones having maternal and paternal chromosomes in different manners to each other.The genetic loci for the disease genes are determined through the conventional processes of positional cloning.Thus, taking advantage of the strategy proposed here, if the abnormality is reproducible using patient-derived pluripotent stem cells, a single carrier of the genetic mutations would be adequate to identify the disease gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan. muto@cdb.riken.jp

ABSTRACT
Forward genetics in humans is beneficial in terms of diagnosis and treatment of genetic diseases, and discovery of gene functions. However, experimental mating is not possible among humans. In order to overcome this problem, I propose a novel experimental procedure to genetically identify human disease gene loci. To accomplish this, somatic cells from patients or their parents are reprogrammed to the pluripotent state, oogenesis is induced, the oocytes are parthenogenetically activated in the presence of cytochalasin, and embryonic stem cells are established from the parthenogenetic blastocysts. This protocol produces a set of diploid pluripotent stem cell clones having maternal and paternal chromosomes in different manners to each other. The genetic loci for the disease genes are determined through the conventional processes of positional cloning. Thus, taking advantage of the strategy proposed here, if the abnormality is reproducible using patient-derived pluripotent stem cells, a single carrier of the genetic mutations would be adequate to identify the disease gene loci.

Show MeSH
Related in: MedlinePlus