Embryoid Body - Based Screen
First Claim
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.
1 Assignment
0 Petitions
Accused Products
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.
-
Citations
20 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. - View Dependent Claims (2, 3, 4, 5, 6)
- ES”
-
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. - View Dependent Claims (8, 9, 10, 11)
- ES”
-
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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
- ES”
Specification