A simple technique for generating probes for RNA in situ hybridization: an adjunct to genome mapping exemplified by the RAG-1/RAG-2 gene cluster.
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.
Boehm
T
T
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Gonzalez-Sarmiento
R
R
Kennedy
M
M
Rabbitts
T H
TH
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins; D009693:Nucleic Acid Hybridization; D051842:Olfactory Marker Protein; D009841:Oligonucleotides / Q000737:chemistry; D015513:Oncogene Proteins; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011995:Recombination, Genetic; D013950:Thymus Gland / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics
D015342:DNA Probes; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; C513044:Lmo1 protein, mouse; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D051842:Olfactory Marker Protein; D009841:Oligonucleotides; C497598:Omp protein, mouse; D015513:Oncogene Proteins; D011506:Proteins; D012333:RNA, Messenger; C499308:Rag2 protein, mouse; D014157:Transcription Factors; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein