Worm Breeder's Gazette 10(2): 66

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In situ Hybridization to Ascaris Embryos

Karen L. Bennett

My laboratory is interested in isolating maternal and early 
embryonic mRNAs which segregate with, or are produced in, the two germ 
line precursor cells of the early nematode embryo.  I propose a final 
assay for putative messages to be in situ hybridization to Ascaris 
embryos.  Because the vitelline membrane of Ascaris is highly 
impermeable, it has been necessary to use frozen 10 m sections of eggs 
(sliced eggs!).  This has worked well for the probes tested to date.  
A 1.0 kb subclone from the 26 S subunit of an Ascaris ribosomal gene (
Bach, et al., (1984) NAR 12-3:1313.) has been put into the SP6 
transcription vector in both orientations.  With the antisense 
transcript all cells of the paraformaldehyde-fixed embryos hybridize 
strongly to the hydrolyzed [35S] RNA probe, while the sense strand 
shows no hybridization above background.  In order to determine the 
resolution of our assay and see if the two germ cells can be 
selectively hybridized, I have also used an RNA probe from the Ascaris 
satellite sequence which is lost from all the somatic lineages and 
retained in the germ line during chromatic diminution (Muller, et al., 
(1982) NAR 10-23:7493.).  With 60 cell embryos (after diminution), the 
satellite probe shows high grain accumulation in the two primordial 
germ cells.  The pattern seen in Ascaris looks much like the antibody 
staining with the anti-P granule antibodies in C.  elegans, the two 
germ cells being covered with silver grains in this case.  The 
sensitivity of the hybridization has yet to be tested, as both the 
ribosomal and satellite probes are abundant in the RNA and germ line 
genomic DNA, respectively.  
Helpful, current resource material on in situ hybridization 
techniques includes: 
Jorgensen, E. M.  and R. L.  Garber.  Pro Mega Notes 10:2-5 (1987).  

Angerer, L. M., et al.  Methods in Enzymology 152:649-661 (1987).