Embryoid Body - Based Screen

• US 20070015210A1
• Filed: 07/09/2006
• Published: 01/18/2007
• Est. Priority Date: 11/24/2004
• Status: Active Grant

• Current Assignee: Geneprotech Incorporated
• Original Assignee: Geneprotech Incorporated
• Inventors: Ezekiel, Uthayashanker
• Granted Patent: US 7,803,619 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms

Abstract

Disclosed are embryoid bodies having a uniform size of approximately 415 nm and comprising genetically modified embryonic stem cells, and methods of making same. The genetically uniform embryoid bodies can be multiplexed as one embryoid body per well in a multiwell format, and used as a high to medium throughput screen for test agents that affect the development and homeostasis of animals, including humans. The genetic modification of the embryonic stem cells is a promoter-report-selection construct that enables the selection and detection of cells of a particular lineage in the EB, to determine the effects of a test agent.

Claims

1. A method for making embryoid bodies having a uniform size, comprising (a) genetically modifying an embryonic stem (“

ES”

) cell to contain a reporter gene and selectable marker gene operably linked to a developmentally regulated promoter, (b) growing the ES cells in the presence of feeder cells, (c) predifferentiating the ES cells by removing the feeder cells, (d) dissociating the ES cells and culturing them in differentiation media, (e) placing approximately 1,000 ES cells into a single vessel in media, wherein an embryoid body (“

EB”

) is produced which has a uniform diameter.

2. The method of claim 1 wherein the uniform diameter is approximately 415 nm±

10%.

3. The method of claim 1 wherein the embryonic stem cell is a murine stem cell.

4. The method of claim 1 wherein the developmentally regulated promoter is a necdin promoter.

5. The method of claim 1 wherein the single vessel is a well in a multiwell plate.

6. The method of claim 1 wherein the reporter gene is lac Z and the selectable marker gene is a puromycin-resistance gene.

7. A system comprising a plurality of embryoid bodies in a plate having multiple wells, the embryoid bodies formed according to steps of (a) growing embryonic stem (“

ES”

) cells in the presence of feeder cells, (b) predifferentiating the ES cells by removing the feeder cells, (c) dissociating the ES cells and culturing them in differentiation media, (d) placing approximately 1,000 ES cells into each individual well of the plate having multiple wells, such that an embryoid body (“

EB”

) is produced which has a uniform diameter; and

wherein (e) one or more embryoid body contains a genetic construct, and (f) a majority of the wells contain a single embryoid body.

8. The system of claim 7 wherein the genetic construct comprises a developmentally-regulated promoter operably linked to a selection marker and a reporter marker.

9. The system of claim 8 wherein the developmentally-regulated promoter is a necdin promoter, the selection marker is a puromycin-resistance gene, and the reporter marker is a lac Z gene.

10. The system of claim 7 wherein the ES cells are murine ES cells.

11. The system of claim 7 wherein the uniform diameter is approximately 415 nm±

10%.

12. A method for screening agents comprising the steps of (a) producing embryoid bodies in a first plate having multiple wells, the embryoid bodies formed according to steps of (i) growing embryonic stem (“

ES”

) cells in the presence of feeder cells, (ii) predifferentiating the ES cells by removing the feeder cells, (iii) dissociating the ES cells and culturing them in differentiation media, (iv) placing approximately 1,000 ES cells into each individual well of the plate having multiple wells, such that an embryoid body (“

EB”

) is produced which has a uniform diameter; and

wherein (v) one or more embryoid body contains a genetic construct, and (vi) a majority of the wells contain a single embryoid body;

(b) transferring EBs from the first plate having multiple wells to a second plate having multiple wells containing at least one agent;

(c) waiting a specified amount of time;

(d) assessing the status of gene expression of the embryoid body;

(e) determining the effect of the agent on the development of the embryoid body; and

(f) assigning an attribute to the agent.

13. The method of claim 12 wherein the uniform diameter is approximately 415 nm±

10%.

14. The method of claim 12 wherein the specified amount of time is from between one day and fourteen days, inclusively.

15. The method of claim 12 wherein the attribute is selected from the group consisting of toxic, non-toxic, morpohogenic, non-morphogenic, and therapeutic.

16. The method of claim 12, wherein the agent is selected from the group consisting of virus, bacteria, chemical, drug, and biological macromolecule.

17. The method of claim 16 wherein the biological macromolecule is a morphogen.

18. The method of claim 16 wherein the chemical is selected from the group consisting of vitamin, enzyme cofactor, representative of a synthetic chemical library, representative of a natural chemical library, an animal-derived chemical, a plant-derived chemical, a whole plant extract, a metabolic intermediate, and a carbon nanoparticle.

19. The system of claim 12 wherein the genetic construct comprises a developmentally-regulated promoter operably linked to a selection marker and a reporter marker, wherein the developmentally-regulated promoter is a necdin promoter, the selection marker is a puromycin-resistance gene, and the reporter marker is a lac Z gene.

20. The system of claim 12 wherein the ES cells are murine ES cells.