Notes

1. DIA is produced by transient expression in COS cells using the method described in ref. 21. Serial dilutions of the medium are tested on ES cells plated in 24-well plates. A 100-fold higher dilution than the minimal dilution required to keep ES cells undifferentiated is typically used. Serum batches are tested for their ability to sustain the growth, differentiation, and viability of ES cells grown at clonal density in the presence and absence of DIA.

2. Electroporation of 108 ES cells normally generates between 200 and 400 G418R colonies. Using the gene trap vector pGT1.8geo (8), we find 50% of colonies stain for Pgal activity. A proportion of the white colonies represents genes expressed at low levels in ES cells, which can be induced on differentiation. The remainder probably represents inactive Pgal fusion proteins, such as secretory molecules, and events where sequences from the 5'-end of Pgal have been removed by endogenous exonucleases prior to insertion into the genome. Using the secretory trap vector pGT1.8TM, we find approx 25% of colonies stain positive for Pgal. Almost all show the secretory pattern of staining in the peri-nuclear compartment of ES cells and in multiple cytoplasmic inclusions, and represent fusions to genes encoding a 5'-signal sequence.

3. To screen for genes induced or repressed under various growth conditions, more than one Experimental plate can be seeded. If the assays are to be conducted over a period of days, the Master plate can be frozen at -80°C (22), allowing the recovery of selected colonies at a later date.

Fig. 3. Arrangement of oligos used to RACE clone genes disrupted by gene trap vector pGT1.8geo (A) and the secretory trap vector pGT1.8TM (B). Both vectors contain the mouse Engrailed-2 (En2) splice acceptor (SA) linked to a Pgeo reporter containing Pgal and neomycin phosphotransferase activities. The secretory trap vector contains a fragment of CD4 that encodes the transmembrane domain (TM). Oligo 1, primer for first-strand cDNA synthesis; oligo 2, primer for second-strand cDNA synthesis; oligos 3 and 4, first-round PCR primers and oligos 3 and 5, second-round PCR primers (see Table 1).

Fig. 3. Arrangement of oligos used to RACE clone genes disrupted by gene trap vector pGT1.8geo (A) and the secretory trap vector pGT1.8TM (B). Both vectors contain the mouse Engrailed-2 (En2) splice acceptor (SA) linked to a Pgeo reporter containing Pgal and neomycin phosphotransferase activities. The secretory trap vector contains a fragment of CD4 that encodes the transmembrane domain (TM). Oligo 1, primer for first-strand cDNA synthesis; oligo 2, primer for second-strand cDNA synthesis; oligos 3 and 4, first-round PCR primers and oligos 3 and 5, second-round PCR primers (see Table 1).

4. Other methods of RNA preparation have recently been shown to work well for RACE cloning. These include acid phenol (RNazol B, Biogenesis Ltd.) and poly dT-based bead selection (Dynabeads, Dynal Ltd.) protocols.

5. Before digesting second-round products, it is advisable to check the fidelity of the preceding reactions. This is done by running 5 pL of second-round PCR products on a 1.5% agarose gel and carrying out Southern blot analysis using a vector probe that includes sequences contained in the RACE products (see Fig. 3). If the reactions have worked, the autoradiograph will require less than a 15-min exposure. In most cases, we see a heterogeneous smear of amplified products. However, if the fusion transcripts are small, a band may be seen.

6. Colonies are selected by digesting plasmid minipreps with the enzymes used for cloning the RACE products and carrying out Southern analysis using the SA probe (see Fig. 3). Colonies can also be screened using a PCR strategy employing primer 5 and a primer in the pBluescript polylinker. Only clones containing gene trap vector sequences should be amplified. We normally recover products that contain between 100 and 700 bp of sequence from the disrupted gene. To confirm that the RACE clone truly represents the disrupted gene, Northern blot analysis should be performed using the RACE clone as a probe. Alternatively, if the RACE clones are small, RNase protection experiments can be used.

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