Haploid human embryonic stem cell lines and somatic cell lines and methods of making the same

Patent metadata

Field	Value
Publication number	US-12091678-B2
Application number	US-202017122562-A
Country	US
Kind code	B2
Filing date	Dec 15, 2020
Priority date	Jul 29, 2015
Publication date	Sep 17, 2024
Grant date	Sep 17, 2024

Abstract

Haploid human embryonic stem cells and cell lines, haploid multipotent human cells, and haploid differentiated human cells are provided. In addition, methods of making and using the haploid human cells are provided.

First claim

The invention claimed is:

1. A method of genetic screening comprising:
a) exposing an enriched population of human haploid embryonic stem (ES) cells capable of differentiating into a terminally differentiated haploid cell, to a mutagen to induce at least one mutation in the cells, wherein said terminally differentiated haploid cell is a haploid mature neuron or a haploid cardiomyocyte;
b) selecting human haploid ES cells in said enriched population that contain said mutation; and
c) identifying a genotypic and/or phenotypic effect of said mutation in said selected human haploid ES cells.

2. The method of claim 1 , wherein said genetic screening is forward genetic screening.

3. The method of claim 1 , wherein said genetic screening is loss-of-function genetic screening.

4. The method of claim 1 , wherein said mutagen is selected from the group consisting of a physical mutagen, a chemical mutagen, and a biological agent.

5. The method of claim 4 , wherein said physical mutagen is ionizing radiation.

6. The method of claim 4 , wherein said chemical mutagen is an alkylating agent.

7. The method of claim 4 , wherein said biological agent is a gene trap vector.

8. The method of claim 4 , wherein said biological agent is selected from a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), and a CRISPR/Cas9 system.

9. The method of claim 8 , wherein said biological agent is a CRISPR/Cas9 system.

10. The method of claim 1 , wherein said selecting is selecting human haploid ES cells in said enriched population that contain said mutation in an expressed locus.

11. The method of claim 10 , further comprising determining said locus comprising said mutation.

12. The method of claim 11 , wherein said determining comprises sequencing of said locus.

13. The method of claim 1 , further comprising determining the genomic location of said mutation.

14. The method of claim 13 , wherein said determining comprises sequencing said genomic location.

15. The method of claim 1 , wherein said phenotypic effect is drug resistance.

16. The method of claim 15 , wherein said identifying comprising contacting said selected human haploid ES cells with said drug and selecting resistant cells.

17. The method of claim 1 , wherein said mutation is an autosomal mutation.

Assignees

• New York Stem Cell Found Inc
• Yissum Res Dev Co Of Hebrew Univ Jerusalem Ltd

Inventors

• Egli Dietrich M
• Benvenisty Nissim
• Sagi Ido

Classifications

• C12N5/0612

  sorting of gametes, e.g. according to sex or motility · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2517/10

  Conditioning of cells for in vitro fecondation or nuclear transfer · CPC title