This study demonstrates a method for co-culturing embryonic cells with reporter cells to analyze retinoic acid signaling during early development. By utilizing a transgenic construct, the expression of beta-galactosidase is monitored as an indicator of retinoic acid release from embryonic cells.
Take a mouse embryo at an early developmental stage immersed in a buffer. Transfer the embryo into an enzyme solution and incubate it under physiological conditions, allowing the enzymes to break down the intercellular connections.
Mechanically dissociate the digested embryo to form a cell suspension.
Transfer the embryonic cells onto an adherent culture of reporter cells.
The reporter cells carry a transgenic construct containing the lacZ gene, which encodes the enzyme beta-galactosidase. The expression of lacZ is regulated by a retinoic acid response element, or RARE.
The embryonic cells release retinoic acid, or RA, a signaling molecule for embryonic development.
RA enters the reporter cells and binds to its receptor complex on the RARE, triggering the formation of a transcriptional activator complex.
The complex activates the expression of lacZ, resulting in the production of beta-galactosidase.
Beta-galactosidase production in the reporter cells indicates the release of RA by the embryonic cells.
For embryonic cell co-culturing, begin dissection of E8.5 embryos. Next, load a new 1.5-milliliter tube with 10 microliters of trypsin, and using a widened-bore P20 pipette tip, transfer the whole embryo into the tube, and place the tube on ice.
After all the embryos have been isolated, incubate all the tubes at 37 degrees Celsius for five minutes to dissociate the tissues. After 5 minutes, gently triturate each tube's contents using P20 pipette tip and transfer 10 microliters to a well of the prepared 96-well plate with F9 RARE LacZ cells. Culture the plate overnight.
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