Worm Breeder's Gazette 11(1): 67
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.
My major interest is to understand the network of regulation of gene expression in embryogenesis of C. elegans. To attack this problem, I am developing a new methodology for isolating genes which are specifically expressed in a stage and/or some cell(s) of the embryo because I think that such genes must play important roles in embryogenesis. For isolation of differentially expressed genes, cDNA- mRNA subtractive hybridization is very powerful. However, conventional subtraction methods need considerable amount of driver mRNA and the driver mRNA should be isolated from cells as closely related as possible to the cells from which cDNA is made. Therefore, I prefer to develop a new method to make a set of cDNA from single embryos of various stages, amplify them in vitro using PCR (polymerase chain reaction) and then perform subtractive hybridization, rather than to deal with a large amount of synchronized embryos. Outline of the method is as follows; (1) Each embryo that had just completed 1st cleavage was picked up, washed, transferred to an agarose pad and incubated at 22 C for an appropriate period. The developmental stage of the embryo was confirmed on a Nomarski microscope. (2) The embryo was transferred onto an inverted lid from a 0.5 ml eppendorf tube and squashed in GuEST (D.Pilgrim,WBG 10(2),154) buffer. (3) Total RNA was extracted twice by PC1 (ibid.) and precipitated by ethanol using glycogen as carrier. (4) cDNA was made on the RNA using 5'-tag1(20mer)-oligo(dT)(12)-3' as primer. In this reaction [32P]-dCTP was used at high specific activity (400 Ci/mmole) and the resulting cDNA was stood for 2 days to shorten about 1 kb by [32P] disintegration. This is to ensure that the amplified cDNA represents the original cDNA, since the efficiency of PCR drops as the size of the target DNA increases. (5) After dG tailing (20-30 mer) by Terminal transferase, the cDNA was amplified by PCR using 5'-tag2(20mer)-oligo(dC)(12)-3' and tag1 as primers. (6) [32P]-labeled antisense-DNA and 10-fold amount of unlabeled sense-DNA were synthesized by further PCR reaction on thus amplified cDNA made from two different stages of embryos, respectively, using only tag1 and tag2 as primer, respectively. They were hybridized using PERT (phenol emulsion reassociation technique) (Kohne et al., Biochem. 16,5329(1977)) to ensure subtractive hybridization even at a low concentration of DNA. (7) The labeled DNA remaining single-stranded was isolated by hydroxyapatite chromotography and used to screen a cDNA library. In a preliminary experiment, I performed two subtractions, namely, 6. 5hr (after 1st cleavage) embryo-3hr embryo and 3hr embryo-2hr embryo. After screening some 5x10+E4 plaques of Ahringer's embryonic cDNA library, I got 5 and 2 very faint signals, respectively. There was no common signal between them, suggesting that the subtraction did work. But the problem is signal intensity. Since an embryo is a mixture of different types of cells, it is anticipated that, even if abundant expression of a gene occurs in some cell(s), the message is still rare in the whole mRNA. Thus, I am developing a method to boost the signal by amplifying the subtracted cDNA using two different sets of primers.