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ES cell lines are thawed and passed for 6 days in ES cell medium in the absence of G418. On the day of injection, the medium should be changed several hours before harvesting the cells. A confluent 25 cm2 flask is trypsinized for 3-4 minutes and diluted into 9 ml of cold ES cell medium without LIF, pelleted, and resuspended in 0.8 ml of ES cell medium (without LIF) in a sterile 1.5 ml screw-top microcentrifuge tube. Before they are added to the injection chamber, cells are kept on ice (for up to several hours) to prevent clumping. Blastocysts are flushed from pregnant C57BL/6 females (Jackson Laboratory) and collected into a CO2-independent medium (Gibco-BRL) containing 10% FBS.
Blastocysts are expanded for 1-2 hours in ES cell medium in a 37°C/6% CO2 incubator, transferred to a hanging drop chamber, and cooled to 4°C. ES cells are added to the hanging drops, and the blastocysts are injected with enough cells (20 or more) to fill the blastocoele. Injected blastocysts are then transferred to pseudopregnant recipient females (10-15 blastocysts/uterine horn). Typically, injection of 10 blastocysts will yield an average of twice male chimeras with germline mosaicism. Of note, the 129/Ola cells carry the recessive pinkeye (p) and chinchilla (cch) mutations; strong chimeras exhibit patches of cream colored fur and can have pink eyes.
§ Excerpted from "Gene Trapping Methods for the Identification and Functional Analysis of Cell Surface Proteins in Mice" by William C. Skarnes in: Methods in Enzymology, volume 328, edited by John N. Abelson, Jeremy Thorner, and Scott D. Emr. Copyright © 2000 by Academic Press. This material may not be reproduced, stored in a retrieval system, or transmitted in any form or by any means without the prior written permission of the publisher.