Worm Breeder's Gazette 13(1): 26 (October 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Isolation of Tc1 insertion mutants from the mutant bank; an update

Ronald H.A. Plasterk, Marianne de Vroomen, Rik C. Korswagen

The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066CX Amsterdam

After the worm meeting 1993 we have received several requests to isolate Tc1 insertion alleles from the frozen library. Marianne de Vroomen has joined the lab, and spends most of her time on the isolation of mutants from the library. This abstract gives an overview of the genes that we have looked at or are looking at. Thus far insertion alleles of most genes can be found; nevertheless we are still expanding the library and improving the technique (see Korswagen et al., Meeting 1993, p. 254).

We have obtained one or more lines with insertions in the following genes:

gpa-1 pgp-1 prk-2 pes-9

gpa-2 pgp-2 ceh-6 lin-26

gpa-3 pgp-3 ceh-13 flp-1

gsa-1 pgp-4 ceh-18 ZK637 .5

goa-1 prk-1 ceh-22 gpb-1


We have (partial) addresses for the following genes (which means that usually -but not always- a clonal line can be obtained):

dynamine crf-2 ncc-1

ZK637 globin gene G luR40 PCTAIRE-kinase

cap-1 SR p30b gqa-1

We have not yet looked through the complete library at the following genes:

SOD (II) ubc-1 lir-1 pes-10

SOD(III) SR p20 kin-15 and kin-16 pes-1

elt-1 apl-1

We have failed to find an insert and will try with new primers for the genes:

ubc-2 cmk-1 cm14 h10 wnt-1

We are still ready to look for insertions in your favourite gene(s). If you want us to do that, please send an E-mail and send primers that follow the guidelines below.

The closer you stick to the guidelines the more searches we can do. One point may need clarification: having only a cDNA sequence one could in theory also design primers, and test them on genomic DNA to check that they do not cross an intron-exon boundary and that the prime sites for the two nested oligonucleotides map close to each other. But it is much better to have at least a few kbp of genomic sequence: that way you are 100% sure of the primer sites, you can later choose primers at the appropriate distance to select deletion derivatives, and you can determine the precise insertion site of Tc1 even when it has integrated into an intron.

Guidelines for primer choice and shipping

To do a search for a Tc1 allele of a sequenced gene we would like to have:

1. a print of 3 kbp of continuous genomic sequence, with the exons of the gene indicated, and the primers indicated (with their direction!). If you have a bit less than 3kbp of sequence information we can do a search nevertheless; the 3kbp is based on our experience that is an ideal distance to choose primers to select deletion derivatives of a Tc1 allele.

2. Two sets of nested primers, each at one end of the 3 kbp, and pointing inward. We like them as follows:

-two primers pointing the same direction, not further apart than necessary (100, 200 bp is fine).

-approx 50% GC, 21-23 nt long, no funny sequences (palindromes, repeats or identical sequences), somewhat balanced composition of all 4 nt, and at the 3' end a G or C.

-Primers in TE, concentration set at 100µM (100µl is plenty). You can send them without dry ice or anything, but perhaps it is good to use Federal Express or an equivalent 24 hour service. -Life is easy for us if you use a simple name for the primers. E.g. the first three letters of your name, plus a number. So e.g. JON I, JON2 , JON3 and JON4 .Then make sure that JON2 is the nested primer of JON I, and JON4 the nested primer of JON3 . Please mark the tubes on the side and on the top, and make sure the label or marking sticks.

In principle one rather than two sets of primers is sufficient, but it gives us an extra option, and you can later use the primers to select deletion derivatives. Also, having two sets of primers at this distance allows you to do a PCR with all 4 combinations to check that the primers are in order (this is not necessary if you know that your oligo-synthesis can normally be trusted).

If you ask for insertions in more than one gene, please indicate your priority.