Use of haploid genomes for genetic diagnosis, modification and multiplication

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

[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.[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.[0004] The diploid number (2n) equals twice the haploid number and is the characteristic number for most cells other than gametes. A zygote is the diploid cell resulting from the fusion of male and female gametes during fertilization. THE DICTIONARY OF CELL BIOLOGY 103, 139, 388 (J. M. Lackie et al., eds. 1995). Only a (diploid) zygote is capable of giving rise to a viable offspring. By con...

AI Technical Summary

Benefits of technology

[0023] By "multiplication" is meant increasing the number of cells comprising the desired haploid genome of male or female origin.

[0048] This is an advantageous aspect of the invention because allelic differences at a locus will not interfere with the desired recombination events. Also, the present invention allows for the same locus to be targeted in both the male and female haploid cell lines, and the resultant modified male and female haploid genomes to be combined to produce a diploid embryo that is homozygous for the particular modification, e.g. deletion of a particular gene.

[0049] As discussed, the invention described herein improves upon prior methods of preimplantation genetic diagnosis (PGD), because these methods do not involve the manipulation of an embryo. Generally, few embryos are available for screening. Moreover, removal of the cells from an embryo for testing can be harmful for further development of the embryo. Often only one or very few cells are available for genetic testing, which can lead to inaccurate results due to DNA loss or DNA contamination. Finally, there are ethical considerations regarding embryo disposal. Genetic screening of haploid DNA offers the advantage that if male and / or female gametes are screened then, even with few gametes, the total possible combination becomes large.

[0050] In the case of sex-linked genetic diseases, screening can be done on sperm only, which is typically easy to obtain in large quantities. If the sperm is not available in large quantities, then multiplication of the sperm genome can also be useful. The technique makes many identical copies of the genome available for screening to minimize the likelihood of misdiagnosis, and permits additional samples to be analyzed for verification of results. The ethical concerns about working with and manipulating sperm are minimal in comparison with those for working with embryos.

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.