2004 West Coast Worm Meeting abstract 95

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.

A novel method for tissue-specific profiling of genes in C. elegans

William J. Michels1,2

1 Biotica Research Corp., 1796 University Avenue, Berkeley, CA
2 correspondence: P.O. Box 12984, Berkeley, CA 94712 biotica@sbcglobal.net

Advances in understanding multi-cellular organisms are encumbered because of inherent problems with cell and/or tissue heterogeneity. Particularly challenging, with regard to studies of tissue-specific gene expression, is the fact that cells and tissues of interest often cannot be isolated away from unwanted cells and tissues. The inability to precisely isolate tissue types can lead to incorrect assignment of gene expression, and to misinterpretation of promoter and gene function. This difficulty slows advances in understanding multi-cellular organisms, such as C. elegans, despite extensive genomic sequence data.

 

To address this problem, SL-addition trans-splicing is engineered to act as a tool for tissue-specific profiling of genes in C. elegans. Synthetic sequences are inserted adjacent to or within the spliced leader mini-exon. Alternatively, the spliced leader mini-exon is mutated. In keeping with the nomenclature exon and intron, the synthetic RNA sequences that are "tagged on" to the 5'-end of genes in a SL-addition trans-splicing reaction are designated "Tagon(TM) sequences", and the genes that donate them as "Tagon(TM)-SLRNA" genes.

 

After Tagon(TM)-SLRNAs are spliced onto pre-mRNAs the Tagon(TM) sequence can be used to purify expressed genes by simple oligonucleotide-mediated hybridization. These isolated mRNAs can be used to generate cDNA libraries, or directly labeled with fluorescent and/or radioactive tags for use as a 'probe' in microarray studies. Alternatively, Tagon(TM) trans-spliced mRNAs can be preferentially cloned by priming second-strand cDNA synthesis in a RACE (rapid amplification of cDNA ends) reaction, using a designed oligonucleotide corresponding to the synthetic Tagon(TM)sequence.

 

In order to enable the recovery of expressed genes subsets from defined tissues and/or cells, a Tagon(TM)-SLRNA gene is cloned downstream of a known cell type specific or tissue-specific structural gene promoter. SL-RNA genes have an internal promoter element that must be obliterated to achieve tissue-specific expression of the Tagon(TM)-SLRNA gene construct.

 

Tagon(TM) profiling can be multiplexed. Because a Tagon(TM) sequence provides a unique nucleic acid "tag" for eventual recovery, Tagon(TM)-SLRNA genes each encoding a unique Tagon(TM) sequence can be cloned and expressed under the control of different promoters. An individual strain can be created containing multiple different Promoter::Tagon(TM)-SLRNA constructs (PromoterX::Tagon(TM)1-SLRNA, PromoterY::Tagon(TM)2-SLRNA, etc.). This technique enables the recovery of different populations of trans-spliced tissue-specific mRNAs from the same animal, and the creation of precisely defined cDNA libraries from C. elegans and other organisms previously refractory to this type of analysis.