Embryoid body-based screen

• US 7,803,619 B2
• Filed: 07/09/2006
• Issued: 09/28/2010
• Est. Priority Date: 11/24/2004
• Status: Expired due to Fees

• Current Assignee: Geneprotech Incorporated
• Original Assignee: Geneprotech Incorporated
• Inventors: Ezekiel, Uthayashanker
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US11/456,203
• Publication Number:
  US 20070015210A1
• Time in Patent Office: 1,542 Days
• Field of Search: 435/325
• US Class Current: 435/377
• 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 mouse embryoid bodies having a uniform size, comprising (a) genetically modifying a mouse embryonic stem (ES) cell to contain a transgene comprising a reporter gene and selectable marker, both operably linked to a developmentally regulated promoter and operably separated by an internal ribosome entry site (IRES), (b) growing the ES cells in the presence of feeder cells and leukemia inhibitory factor (LIF), (c) removing the feeder cells, (d) dissociating the ES cells in differentiation media, (e) placing approximately 1,000 dissociated ES cells of step (d) in a 200 μ

l volume into each well of a plurality of wells of a polyvinyl carbonate multiwell plate in media, wherein the multiwell plate is not subjected to centrifugation and a single mouse embryoid body (EB), that has a uniform diameter relative to other EBs formed in, is produced per well in at least 92% of the wells.

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

10%.

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

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

5. A system comprising a plurality of embryoid bodies in a polyvinyl carbonate plate having multiple wells, the embryoid bodies formed according to steps of (a) introducing a genetic construct into a mouse embryonic stem (ES) cells, (b) growing said ES cells in the presence of feeder cells and LIF, (c) removing the feeder cells, (d) dissociating the ES cells in differentiation media, (e) placing approximately 1,000 ES cells in a 200 μ

l volume into each individual well of the plate having multiple wells, wherein the plate is not subjected to centrifugation such that embryoid bodies (EB) are produced that have a uniform diameter relative to other EBs formed in other wells; and

wherein (f) one or more embryoid body contains a genetic construct, and (g) at least 92% of the wells contain a single embryoid body, wherein the genetic construct comprises a developmentally-regulated promoter operably linked to a selection marker and a reporter marker that are operably separated by an IRES.

6. The system of claim 5 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.

7. The system of claim 5 wherein the uniform diameter is approximately 415 nm±

10%.