Electroporation and isolation of ES cell lines^§

Electroporation is the preferred method for transfecting ES cells with plasmid-based gene-trap vectors because the concentration of DNA and cell density can be adjusted to favor single-copy insertions. The conditions described below have been optimized for feeder-independent ES cells to maximize colony number and minimize multicopy insertions. Occasionally, multiple copies of the vector will integrate at a single site, but these multicopy insertions do not appear to interfere with 5' RACE (Rapid Amplification of cDNA Ends), reporter gene expression, or rates of mutation.

DNA preparation

Vector DNA may be isolated by alkaline lysis and cesium chloride density centrifugation or by Quiagen column purification. Allow two days before the electroporation for the preparation of the DNA sample.

1. Linearize the vector by digesting 150 µg of pGT1.8TM DNA overnight at 37°C with HindIII restriction enzyme (0.5 U/µg) in a final volume of 0.3 ml.
2. Precipitate the DNA sample with two volumes of absolute ethanol on ice for 5 minutes. Spin down the DNA in a microcentrifuge and wash pellet several times with 70% ethanol.
3. Drain off as much 70% ethanol as possible and allow the remainder to evaporate in a sterile laminar flow hood, leaving the lid of the tube open (1 hour is usually sufficient).
4. Resuspend DNA in 0.1 ml of sterile PBS and vortex occasionally over a period of 4 hours or more to ensure that the DNA is in solution.

Electroporation of ES cells

For optimal results, the ES cells should be approximately 80% confluent on the day of electroporation, and the medium should be changed a few hours before the cells are harvested.

1. Trypsinize the contents of three 175 cm² flasks, (5 ml of trypsin/flask). Add 10 ml of medium to each flask and combine the cells in a 50 ml sterile centrifuge tube. Pellet cells for 5 minutes at 1200 rpm and resuspend in 20 ml PBS.
2. Count cells, repellet, and resuspend the cells at a concentration of 1 × 10⁸ cells/0.7 ml of PBS.
3. Add 0.7 ml of cells to the tube containing 0.1 ml of linearized vector DNA, quickly mix with a 1 ml plastic pipette and transfer immediately to a 0.4 cm electroporation cuvette. Electroporate (Bio-Rad Gene Pulser unit) at 3 microF/800V (time constant = 0.04 msec). With the BioRad GenePulser XCell unit, use the Time Constant Protocol at a setting of 0.2 msec and 800 volts. After pulsing, the results screen should indicate a capacitance of 10 microF and an infinite resistance.
4. Allow cells to recover in the cuvette for 20 minutes at room temperature and then transfer them to 200 ml of ES cell medium. Plate 10 ml (5 × 10⁶ cells) onto each of 20 gelatinized 10 cm diameter tissue-culture petri dishes.
5. Aspirate medium the following day and replace with medium containing 125 mg/ml Geneticin. Change the medium daily for the first 4-5 days. Once most of the cells have been cleared from the plate, the medium may be replaced on alternate days. Eight days after electroporation, the colonies should be about 1 mm in diameter and are ready to be picked.

Picking G418-resistant colonies and replica-plating

1. Count and circle colonies on the bottom of each dish with a marker pen. Gelatinize the appropriate number of 48-well plates.
2. Aspirate culture medium and add 10 ml of room temperature PBS to each dish. If possible, colonies should be picked in a laminar flow hood to minimize contamination. Using a Pipetman (P200) and sterile plugged (aerosol-resistant) tips, break up the colony and collect in 50 µl of PBS. Transfer the cell clumps to a 48-well plate and repeat until all the wells contain cells.
3. Add 50 µl of a 2× trypsin solution to each well and incubate for 10 minutes at 37°C. Tap plates to disperse the cells into small clumps and add 1 ml of medium to each well. The following day, replace with 0.5 ml of medium.
4. When the cells approach confluence, trypsinize with 100 µl of 2× trypsin for 2-4 minutes and then add 1 ml of medium. Using a Pipetteman (P1000) and sterile plugged tips, transfer 0.4 ml to two sets of gelatinized 48-well plates (replica plates for X-gal staining and PLAP staining) and 0.2 ml to a third set of gelatinzed 48-well plates (master plate for expanding cell lines). Add 1 ml of medium to each well of the master plate. (Note: Individual clones grow at different rates, so cells are harvested in batches over a period of 1 week. As a rule, we replenish the medium at least every 2 days until the wells reach near-confluence.)
5. One day after plating, the replica plates are stained with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) or NBT/BCIP staining solutions to detect beta-gal or PLAP (human placental alkaline phosphatase) activity, respectively, and incubated overnight.
6. On the following day, each of the X-gal stained wells are examined under bright-field illumination for cell lines that display the "secretory" pattern of beta-gal localization: peri-nuclear staining and multiple cytoplasmic dots as opposed to a single dot of expression commonly seen (see ref. 24 for examples of the secretory staining pattern). The secretory pattern can be seen best in the subset of flattened, spontaneously differentiated cells in the culture. It is more difficult to distinguish the pattern in lines where beta-gal expression is confined to undifferentiated ES cells. Roughly half of the beta-gal positive clones should display this pattern.
7. Cell lines that display the secretory pattern of beta-gal staining are selected from the master 48-well plates and expanded for RNA isolation and for storage in liquid nitrogen. Typically, cells in confluent 48-wells are expanded up to 12-well plates and then split into duplicate 6-well dishes, one for RNA, one for freezing. It is important to maintain G418 selection during expansion to prevent wild-type (non-transfected) cells from contaminating the cultures.

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§ 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.