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Protocols: ES Cell Culture - Gene Targeting - Protocols

Isolation of Primary Fibroblasts from Mouse Embryos

	1.	Treat 10 cm tissue culture plates with 0.1% gelatin 
for at least two hours before use.

	2.	Sacrifice the pregnant female mouse (day 13 or 14 p.c.) 
by cervical dislocation.  Dissect out the uterine horns and place into 
a petri dish containing PBS.

	3.	Separate each embryo from its placenta and surrounding 
membranes.  If desired, keep the yolk sac for genotyping.

	4.	Wash each embryo by transferring it to a petri dish containing 
clean PBS.

	5.	Using a Pipetman with the end of the tip snipped off, 
transfer the embryo into the barrel of a 1cc syringe fitted with an 
18G 11/2" needle and, using the syringe's plunger, force the embryo 
through the needle into a sterile 15 ml tube containing 1 ml of medium 
(DMEM, 1X GPS, and 15% FCS).

	6.	Working in the laminar flow hood, aseptically transfer the 
1 ml of embryo cells to a sterile 15 ml tube containing 10 ml of medium 
(DMEM, 1X GPS, and 15% FCS).

	7.	Aspirate the gelatin from the 10 cm plates and plate out the 
suspension of embryonic cells.

	8.	Incubate the plates @ 37o C until the cells have reached 
confluence (approx. 5 days).  (The primary fibroblasts will be the only 
cells that attach and proliferate.)

	9.	At confluence, split the plate 1:3 by doing the following:

	a.	Wash the plate twice with PBS.

	b.	Add 2 ml of trypsin per 10 cm dish and incubate @ 37o C 
            for 7 minutes.

	c.	Add 5 ml of medium (DMEM, 1X GPS, and 10% FCS) to the 
            plate to neutralize the trypsin.  Resuspend the cells by 
            pipetting up-and-down several times.

	d.	Uniformly divide the cells among three 10 cm gelled plates, 
            each containing 8 ml of medium (DMEM, 1X GPS, and 10% FCS).  
            These plates are designated as Passage No. 1.

	10.	Upon confluence, harvest and freeze the primary fibroblasts 
@ a freezing density of 3.0 x 107 cells/ml.  Reserve a fraction of the 
cells to seed a 6 cm gelled dish from which DNA will be prepared for 
genotyping (unless the yolk sac was used for genotypic analyis).  
The frozen cells are designated as Passage No. 2.