Worm Breeder's Gazette 11(3): 12
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.
We previously reported that when inflating gonads with the rol-6 plasmid (WBG 11-1:18) the efficiency of transformation was saturated at concentrations above 20 g/ml of pRF4. However in those experiments we were coinjecting a second plasmid at 50 g/ml and we had not anticipated that the added DNA would alter the efficiency of transformation. Since then we have found that added DNA can dramatically alter the outcome of transformation experiments. For example: 1) Injection of sup-7(st5) (on pAST 19, provided by A. Fire) yielded healthy suppressed lines carrying a low copy number of pAST 19 on extrachromosomal arrays but only when injected at 1 /ml along with a 100 fold excess of a second plasmid. 2) Homology between coinjected sequences correlated with a uniform incorporation into co-arrays, non- homology correlated with a more haphazard incorporation. And 3) The injection of single stranded oligos along with double stranded DNA led to low copy integrative transformation (WBG 11-2:28). In order to examine the affect of pRF4 concentration on the frequency of transformation (when injected alone) serial dilutions were made in a range from 200 to 12.5 g/ml and were injected into n1270e187 animals (this putative null allele of rol-6 is essentially wild type in appearance and behavior but yields two to three times as many F1 rollers and lines as N2). The average number of F1 rollers per injected animal (and the maximum number from any single injected animal) increased three to four fold as the DNA concentration was increased from 12.5 to 50 g/ml (Table 1). Above 50 g/ml the frequency of F1 rollers appeared to be saturated. The germline transformation efficiency (total lines and lines total F1) showed a more dramatic dependence on DNA concentration. From 391 F1 rollers obtained from injections of 25 g/ml (or less) of pRF4 only one germline transformant was found. F1 rollers obtained at 100 and 200 g/ml were roughly four times more likely to give rise to rolling progeny than were rollers obtained at 50 g/ml. There was no correlation between the number of rollers produced in the F1 by a given injected individual and the probability that those rollers would breed true. Thus the DNA concentration and not the quality of the injection appeared to govern the frequency of heritable transformation. Some DNAs when coinjected with pRF4 consistently reduced while others consistently increased the frequency at which F1 rollers gave rise to heritably transformed lines. One plasmid which appeared to have a strong positive affect on the frequency of germline transformation was an actin-4 -galactosidase construct in pBR322 ( provided by Jocelyn Shaw). When coinjected with pRF4 at 100 g/ml each, a greater than two fold increase in the number of transformed lines was observed (Table 1). We do not know how coinjected sequences influence the frequency of heritable transformation, presumably they may directly affect the formation of arrays or they may exert a secondary affect by influencing the replication, partitioning or gene expression of the transforming DNA. The sensitivity of germline transformation to the concentration of injected DNA is consistent with assembly of arrays from the injected material by recombination (rather than by replication) and suggests that the size or number of structures assembled (or acquired) by germ nuclei may be important in determining heritability. It is interesting that F1 expression and germline transformation show different kinetics for saturation. It appears as though increasing the pRF4 concentration above 50 g/ml does not lead to an increase in the number of germ nuclei which take up and express the injected DNA, but may cause an increase in the amount of DNA acquired by each germ nucleus. These data suggest that either there are a limited number of nuclei receptive to DNA uptake in the syncytial gonad, or that some other factor(s) exist in the gonadal cytoplasm which limit distribution of the injected DNA to only a subset of the nuclei. [See Figure 1]