Use of haploid genomes for genetic diagnosis, modification and multiplication

• US 8,551,705 B2
• Filed: 01/28/2011
• Issued: 10/08/2013
• Est. Priority Date: 11/02/1999
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Moreira, Pedro, Robl, James M
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/015,968
• Publication Number:
  US 20110287429A1
• Time in Patent Office: 984 Days
• Field of Search: 435/6.12, 435/6.1, 435/354
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer

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.

Claims

1. A method for selecting an animal cell comprising a haploid genome, comprising the following steps:

(i) producing an animal embryo comprising a haploid genome of male or female origin;

(ii) culturing said embryo to produce a multi-celled embryo comprising a haploid genome of male or female origin;

(iii) isolating one or more cells from said multi-celled embryo and screening said one or more cells for a desired genetic makeup; and

(iv) selecting one or more cells determined to comprise the desired genetic makeup.

18. The method of claim 1, wherein said screening said one or more cells for a desired genetic makeup in step (iii) comprises one or more methods selected from the group consisting of:

detection of restriction fragment length polymorphisms;

detection of variable number of tandem repeat sequences;

detection of dinucleotide repeat sequences;

detection of short tandem repeat sequences;

single-strand conformational polymorphism analysis;

denaturing gradient gel electrophoresis;

mismatch cleavage analysis by enzymatic or chemical means;

polymerase chain reaction;

rhesus blood group typing;

primed in-situ labeling;

in-situ hybridization;

fluorescence in situ hybridization;

multiplex polymerase chain reaction; and

DNA sequencing.

19. The method of claim 1, wherein said screening said one or more cells for a desired genetic makeup in step (iii) comprises detecting the presence or absence of a specific DNA associated with a phenotype, disease or condition.

20. The method of claim 1, wherein said screening said one or more cells for a desired genetic makeup in step (iii) comprises in situ hybridization, polymerase chain reaction, nested polymerase chain reaction, fluorometric detection methods, restriction fragment length polymorphisms (RFLP) analysis, variable number of tandem repeat (VNTR) detection methods, short tandem repeat (STR) detection methods, single-strand conformational polymorphism (SSCP) analysis, denoting gradient gel electrophoresis (DGGE), and mismatch cleavage analysis.

2. A method for producing an animal embryo having a desired genetic makeup, the method comprising:

(i) producing an animal embryo comprising a haploid genome of male or female origin;

(ii) culturing said embryo to produce a multi-celled embryo comprising a haploid genome of male or female origin;

(iii) isolating one or more cells from said multi-celled embryo and screening said one or more cells for a desired genetic makeup;

(iv) selecting said one or more cells determined to comprise the desired genetic makeup; and

(v) transferring the nucleus of said one or more cells selected in step (iv) into an oocyte and activating said oocyte to produce an embryo comprising said desired genetic makeup.

3. The method of claim 2, wherein said oocyte is an enucleated oocyte or a haploid oocyte.

4. The method of claim 3, further comprising introducing a second haploid genome into said enucleated oocyte.

5. The method of claim 3, wherein said haploid oocyte comprises a haploid genome having a desired genetic makeup.

7. The method of claim 2, wherein said animal embryo is produced by activation of an oocyte in which half of the chromosomes are extruded in the polar body.

8. The method of claim 2, wherein said animal embryo is produced by fertilization of an oocyte and removal of a male pronucleus therefrom.

9. The method of claim 2, wherein said animal embryo is produced by activation of an oocyte to provide an oocyte containing two female pronuclei, and removal of one of said pronuclei.

10. The method of claim 2, wherein said animal embryo is produced by insertion of a diploid cell nucleus into an immature oocyte followed by separation of said chromosomes into two haploid nuclei.

11. The method of claim 2, wherein said animal embryo is produced by transfer of the nucleus of a parthenogenetic cell into an enucleated oocyte.

12. The method of claim 2, wherein said animal embryo is derived from a fertilized oocyte from which the female pronucleus is removed.

13. The method of claim 2, wherein the animal embryo is derived by fertilization of an enucleated oocyte.

14. The method of claim 2, wherein the animal embryo is produced by artificial de-condensation of a sperm nucleus which is then injected into a non-oocyte derived cytoplast.

15. The method of claim 2, wherein said animal embryo is human.

16. The method of claim 3, wherein said oocyte is a haploid oocyte and wherein said embryo comprising said desired genetic makeup is diploid.

17. The method of claim 2, wherein said haploid genome is genetically modified.

6. A method for producing an animal embryonic stem cell having a desired genetic makeup, the method comprising:

(i) producing an animal embryo comprising a haploid genome of male or female origin;

(ii) culturing said embryo to produce a multi-celled embryo comprising a haploid genome of male or female origin;

(iii) isolating one or more cells from said multi-celled embryo and screening said one or more cells for a desired genetic makeup;

(iv) selecting said one or more cells determined to comprise the desired genetic makeup;

(v) transferring the nucleus of said one or more cells selected in step (iv) into an oocyte and activating said oocyte to produce an embryo comprising said desired genetic makeup;

(vi) culturing said embryo comprising a desired genetic makeup produced in step (v) to a stage where the embryo comprises a discernible inner cell mass; and

(vii) isolating said inner cell mass and culturing said inner cell mass, thereby producing embryonic stem cells comprising a haploid genome of male or female origin.