Abstract:
:Two major problems have to be solved in studies of genes near breakpoints of chromosome abnormalities and in large-scale genomic mapping projects: (i) the identification of genes within the large amount on nontranscribed DNA and (ii) the determination of the tissues in which the identified genes are transcribed. In situ hybridization to mRNA is ideally suited to assess gene expression in all tissues but probe preparation presents major difficulties for adapting the technique for rapid screening. Here, we present a procedure to easily generate strand-specific DNA probes for in situ hybridization. In this method, a DNA fragment to be tested in uniformly labeled, denatured, and prehybridized to an excess of competitor single-stranded DNA corresponding to either positive or negative strands of the test fragment. No sequence information is needed. The prehybridized mixture is used directly for hybridization to whole embryo or tissue sections. We demonstrate the utility of this approach for any nonrepetitive fragment by using cDNA probes, intronless genomic probes, or genomic probes comprising transcribed and nontranscribed DNA. As an example, we show that mRNA for the recombination-activating genes (RAG) RAG-1 and RAG-2 is found in thymus of dE16 mouse embryos. Within the thymus, high levels of expression of RAG-1 and RAG-2 are detectable in the cortex but not in the medullary region. This supports the view that RAG-1 and RAG-2 expression is associated with cells known to actively rearrange antigen receptor loci.
journal_name
Proc Natl Acad Sci U S Aauthors
Boehm T,Gonzalez-Sarmiento R,Kennedy M,Rabbitts THdoi
10.1073/pnas.88.9.3927subject
Has Abstractpub_date
1991-05-01 00:00:00pages
3927-31issue
9eissn
0027-8424issn
1091-6490journal_volume
88pub_type
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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更新日期:2001-04-10 00:00:00