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Use of haploid genomes for genetic diagnosis, modification and multiplication

a technology of haploid genomes and genomes, applied in the field of propagation and use of haploid genomes, can solve the problems of dangerous to the developing fetus, aborted or gestated to term, and mammalian embryos have not been used for genetic analysis, etc., and achieve the effect of increasing the number of cells

Inactive Publication Date: 2008-04-10
UNIV OF MASSACHUSETTS REPRESENTED BY ITS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021] By “multiplication” is meant increasing the number of cells comprising the desired haploid genome of male or female origin.

Problems solved by technology

While it has been well reported that mammalian embryos may result from haploid genomes, such mammalian embryos have not been used for genetic analysis.
However, in utero genetic diagnosis is invasive and can be dangerous to the developing fetus (e.g., amniocentesis and chorionic villi sampling).
Fetuses diagnosed with disease can either be aborted or gestated to term, as in utero surgery and gene therapy are still highly risky and experimental.
Thus, based on the foregoing, it is evident that although research is ongoing in perfecting preimplantation genetic screening, as well as manipulation of embryos created in vitro, little progress has been achieved in the genetic screening of gametes or the genetic manipulation of gametes to be used to make transgenic animals.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example

Production of a Haploid Cell Line

Production of Large Murine A9 Cells

[0085] Murine A9 cells (HPRT-) are cultured in 3.75 μg / ml cytochalasin B (Sigma, location) in alphamem (Biowhittaker, location) supplemented with 10% fetal bovine serum for 96 hrs. Cytochalasin B is an inhibitor of microfilaments and will prevent the cells from undergoing cytokinesis while allowing the cell to synthesize DNA and increase in size. After 24 hrs recovery from the drug, cells can be removed from the culture surface and manipulated. Resulting cells are approximately 30 μm in diameter.

Education

[0086] Round glass discs, approximately 2.5 cm in diameter are coated with poly-D-lysine. Cytochalasin B treated A9 cells are plated at 60-80% confluency on the discs and allowed to adhere for 24 hrs. Discs are placed cell-side down in centrifuge tubes containing 5 ml enucleation medium (phosphate buffered saline, 10% fetal bovine serum, 10 μg / ml cytochalasin B). Cells are incubated for 20 min at 37° C. Centri...

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Abstract

Methods for propagating haploid genomes of male or female origina and genetic screening and modification thereof are provided. These haploid genomes may be used to produce haploid embryos, and embryonic stem-like cells and differentiated cells. Also, these haploid genomes and cells containing, may be used as nuclear transfer donors to produce diploid nuclear transfer units. These diploid NT units e.g., human NT units, may be used to obtain pluripotent cells and differentiated cells and tissues.

Description

GOVERNMENT RIGHTS [0001] The invention was developed as a result of the expenditure of funds received from the United States Department of Agriculture and accordingly the government has rights to this invention.FIELD OF THE INVENTION [0002] This invention relates to the propagation and use of haploid genomes for purposes of (1) genetic diagnosis, (2) genetic selection and (3) genetic modification. The selected haploid genomes are useful for the production of embryos and embryonic stem cells when combined with another haploid genome, preferably one having a desired genetic makeup. BACKGROUND OF THE INVENTION [0003] Gametes are specialized haploid cells (e.g., spermatozoa and oocytes) produced by meiosis and involved in sexual reproduction. By contrast, diploid cell has its chromosomes in homologous pairs, and has two copies of each autosomal genetic locus. The diploid number (2n) equals twice the haploid number and is the characteristic number for most cells other than gametes. A zyg...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N15/00C12N5/06C12N5/10G01N33/53C12N15/09C12N15/873C12Q1/02G01N33/566
CPCC12N15/873C12N2517/04C12N2510/00
Inventor ROBL, JAMES M.MOREIRA, PEDRO
Owner UNIV OF MASSACHUSETTS REPRESENTED BY ITS