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Search by in vitro Reporter Expression Pattern

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Expression Trapping

Expression trapping couples gene trapping with the in vitro and in vivo differentiation potential of ES cells. In addition to the ability to develop in vivo into all the tissues of the mouse, ES cells can develop in vitro into a variety of cell lineages dependent upon the culture conditions. We have modified numerous ES cell in vitro differentiation assays into high throughput screens. Thus, gene trap clones are grown in various in vitro differentiation assays, then stained for lacZ expression to determine an expression profile of the "trapped" gene.

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The gene trap vector is introduced into ES cells by electroporation and clones containing gene trap insertions are selected by G418 (neomycin) resistance. NeoR clones are then picked into 96-well master plates which are later replicated to generate frozen stocks for long-term storage of clones, cell lysates for RNA isolation and RACE, and to assay reporter expression in the expression screens developed around the in vitro differentiation assays described below.

In Vitro Differentiation Assays

The following in vitro differentiation assays were used as primary screens to identify clones by potential expression patterns in vivo. To verify the particular lineage in which the reporter gene is expressed, secondary screens should be performed. One of the primary assays used in the screening is the attached embryoid body (EB) culture system. In the absence of leukemic inhibitory factor (LIF), ES colonies spontaneously differentiate into EBs in suspension culture. The complex structure of the EB contains all three germ layers and morphologically resembles the extra-embryonic yolk sac. When EBs are allowed to attached to tissue culture plates, the visceral endoderm layer grows beneath the mesoderm and the EBs become accessible to observation and experimental manipulation. Many tissue types develop in attached EBs including blood islands containing hematopoietic and vascular endothelial cells. Also, EBs contain beating cardiomyocyte foci. In addition to specific lineages assayed such as cardiomyocytes, the attached EB culture system is also used in our screening system to assay clones responsive to retinoic acid, TGF-β proteins, hypoxia, and ionizing radiation. An example of a gene trap insertion into a gene expressed by hematopoietic and vascular cells within a blood island is shown below.

Legend: LacZ, which is denoted by the blue staining, is expressed in the blood island ("bi") and some of the associated vascular endothelium (arrows) in attached EB culture. Notice the red color of the red blood cells and the individual cells within the circulating vascular plexis.

Another in vitro differentiation assay that we use for screening gene trap clones is OP9 co-culture. When ES cells are grown on OP9 stromal cells, ES cells differentiate into mesodermal colonies, which when replated can differentiate into hematopoietic cells. An example of a gene trap insertion into a gene expressed by primitive mesodermal colonies and hematopoietic cells is displayed below.

Legend: LacZ is expressed within a portion of the cells within the mesodermal colonies ("M") shown in the left panel. The right panel shows lacZ expression in a subset of hematopoietic cells (arrows) that differentiated from the mesodermal colonies.

Culture of ES cells on collagen type IV promotes endothelial and vascular smooth muscle differentiation. An example of lacZ reporter gene expression (blue cells) by ES-derived endothelial cells differentiated on collagen IV is shown below.

By culturing ES cells in a serum-free, defined media, ES cells can differentiate into a sphere of cells, known as a neurosphere, that detach from the tissue culture plastic. The neurosphere contains neural stem cells. When the neurospheres are transferred to specific culture conditions, the neurospheres will begin to differentiate into astrocytes, oligodendrocytes, and neurons. Two examples of gene trap insertions into genes expressed by neurospheres are demonstrated below.

Legend: The left panel shows the lacZ fusion protein is expressed in some cells within the neurospheres derived from this gene trap clone. However, the K18F2 gene trap clone shown in the right panel demonstrates lacZ expression by essentially all cells within neurospheres.


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