WO2003062449A9 - Procedes d'isolement de cellules specifiques de caenorhabditis elegans et d'obtention d'informations genomiques a partir de celles-ci - Google Patents

Procedes d'isolement de cellules specifiques de caenorhabditis elegans et d'obtention d'informations genomiques a partir de celles-ci

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WO2003062449A9
WO2003062449A9 PCT/US2003/001632 US0301632W WO03062449A9 WO 2003062449 A9 WO2003062449 A9 WO 2003062449A9 US 0301632 W US0301632 W US 0301632W WO 03062449 A9 WO03062449 A9 WO 03062449A9
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cells
mec
genes
elegans
cell
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PCT/US2003/001632
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WO2003062449A3 (fr
WO2003062449A2 (fr
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Martin Chalfie
Charles Ma
Yun Zhang
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Univ Columbia
Martin Chalfie
Charles Ma
Yun Zhang
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Publication of WO2003062449A2 publication Critical patent/WO2003062449A2/fr
Publication of WO2003062449A3 publication Critical patent/WO2003062449A3/fr
Publication of WO2003062449A9 publication Critical patent/WO2003062449A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the present invention relates to methods of separating one or more particular cell types from the nematode Caenorhabditis elegans, as well as to methods for identifying cell type specific genes therefrom, and analyzing the identified gene sequences to retrieve cell type specific expression elements.
  • C. elegans develops from a single, fertilized cell into an adult animal having complex tissues and organ systems.
  • a substantial proportion of the cells (302 of the 959 somatic nuclei of the adult hermaphrodite worm) constitute a nervous system that can detect odor, taste, and respond to temperature and touch. Because the animal is virtually transparent, its bodily events can be observed under a microscope.
  • C. elegans carries out many of the same processes that humans do, including embryonic development, digestion, reproduction, and senescence, and, as such they are "particularly useful for studying early development, neurobiology, and aging” (Id.). "In fact, every connection in the worm's nervous system has been mapped, and the lineage of each cell in the adult animal's body has been tracked from the moment of fertilization" (Id.).
  • the C. elegans genome contains 19,099 protein-coding genes - about one every 5,000 DNA bases - and 800 or so genes that have other functions, several times the number of genes predicted by classical genetics experiments, approximately 40 percent of which match those of other organisms, including humans (http ⁇ /w ⁇ vw.nhgri.nih.gov/NEWS/Worm/ ⁇ wpr ⁇ ).
  • Nematodes are important pathogens of plants and humans, creating substantial agricultural and medical problems.
  • Currently available nematicides tend to be environmentally and physiologically toxic. It is therefore desirable to develop safer nematode control strategies through a better understanding of nematode molecular biology.
  • the present invention relates to methods for isolating specific types of cells from the nematode Caenorhabditis elegans, isolating RNA therefrom and amplifying the collected RNA to produce amplified nucleic acid, exposing the amplified nucleic acid, under hybridization conditions, to a collection of reference nucleic acids representative of a plurality of nematode genes, and identifying the particular reference nucleic acids which hybridize.
  • the reference nucleic acids that hybridize more strongly, particularly those that exhibit greater hybridization relative to control hybridization levels obtained using nucleic acid collected from other C. elegans cells or the general population of C elegans cells may represent genes that are selectively expressed in the specific cell type isolated.
  • the nucleic acid sequences of the flanking regions of the cell type specific genes isolated may be analyzed to identify cell type specific expression elements.
  • the present invention is based, at least in part, on the discovery that touch receptor neurons of C. elegans could be isolated by engineering the general cell population to contain a nucleic acid construct comprised of a detectable gene product (in the example provided, Green Fluorescent Protein) operably linked to a touch cell-specific promoter and then sorting dissociated cells of the general population based on the presence or absence of the detectable gene product (in the working example, using fluorescence-activated cell sorting). Additional specificity could be obtained by further sorting the cells by other characteristics, such as by size and/or viability.
  • a detectable gene product in the example provided, Green Fluorescent Protein
  • the methods of the invention provide a means for identifying and isolating genes that are expressed at low levels in the general cell population of C. elegans, but at higher levels in specific cell types. Analysis of RNA collected from whole organisms may fail to identify such genes. Accordingly, the present invention provides the capability of identifying genes present in specific cell types which previously would have been virtually unretrievable using present technology. The genes recovered may then be used to identify cell type specific elements. The cell type specific genes identified may then be used to produce model systems of physiologically important functions. The working examples provided here show that genes associated with touch sensitivity were identified. Some of these genes have been found to be associated into ion channels that are responsive to agents that have physiologic activity in humans.
  • the methods of the invention may be used to produce screening systems for human drug development.
  • the usefulness of C. elegans as a screening system for biologically active agents relevant to either pest control in agriculture or human medicine is well known; the present invention provides a substantial enhancement of the specificity of such screening systems .
  • Figure 1 A wild-type touch receptor neuron and a mec-3 mutant cell after 12 hours in culture. Both cells fluoresce because of expressed GFP.
  • the wild-type cell has a single neurite, whereas the mec-3 cell has several shorter neurites.
  • FIG. 1 Northern analyses of amplified RNA from sorted wild-type touch cells (WI and W2), sorted mec-3 mutant cells (Ml and M2), and unsorted wild-type cells (U). Samples were probed with cDNAs for the indicated genes.
  • FIG. 3A-B The MEC-3 ::UNC-86 binding site.
  • A The consensus sequence derived from known MEC-3 ::UNC-86 binding sites from the mec-3, mec-4 and mec-7 genes in C. elegans, C. briggsae and C. vulgaris. The sequence is displayed from 5' to 3'; a perfect match would have a score of two bits.
  • B Positions of significant matches (P ⁇ 10 "4 ) to the consensus sequence in the 5' UTRs of newly identified and known mec- 3-dependent genes. The position of the start ATG is taken as 0; the heights of the bars are proportional to the extent the identified sites match the consensus sequence. The bars under the lines represent the sequences in the other strand.
  • FIG. 4A-F Examples of newly identified r ⁇ .ec-3-dependent genes.
  • SEQ LD NO:l ClustalW alignment of MEC-17 (F57H12.7) (SEQ LD NO:l) and similar proteins: W06B11.1 (SEQ TD NO:2) from C. elegans, CG3967 (SEQ LD NO:3) and CG17003 (SEQ TD NO:4) from D. melanogaster, BAB29231 (SEQ ID NO:5) and BAB22194 (SEQ ID NO:6) from mice, and FLJ13158 (SEQ ID NO:7) and BAB14472 (SEQ ID NO:8) from humans (BAB names are GenBank accession numbers; others are gene names). Amino acids identical (red) or similar (blue) to those in MEC-17 are indicated.
  • the amino acids identical (red) or similar (blue) to those in MEC-17 are indicated.
  • nucleotide changes for the mutations are CTT (L) -> TTT (F) and GAT (D) - GGT
  • FIG. 5A-C Purification of C. elegans neurons by fluorescence-activated cell sorting (FACS). Representative flow cytometric data used for purifying GFP-positive neurons. Data collected on 100,000 total events employing a fluorescence threshold trigger on the cell sorter. All parameters measured using logarithmic signal processing electronics.
  • FACS fluorescence-activated cell sorting
  • Representative flow cytometric data used for purifying GFP-positive neurons. Data collected on 100,000 total events employing a fluorescence threshold trigger on the cell sorter. All parameters measured using logarithmic signal processing electronics.
  • A Forward Scatter laser light (cell size) versus propidium iodide fluorescence used to select viable cells as determined by propidium iodide exclusion.
  • B Green (515-545 mn) versus orange (564-586 nm) fluorescence distribution gated on viable cells was used to select GFP positive cells.
  • C Light scatter distribution of GFP-positive neurons showing heterogeneity of cell size and morphology.
  • GFP-positive neurons are enriched 100 fold by fluorescence-activated cell sorting.
  • the present invention provides for methods for isolating specific cell types of C. elegans or other nematodes.
  • Cell types which may be isolated in this manner include muscle, skin, gut, excretory, germ line, and somatic gonad cells and 118 classes of neurons (see White et ah, 1986, Proc. Roy. Acad. Sci. 314:1-340).
  • a preferred nonlimiting embodiment, in which touch receptor cells are isolated, is set forth in the examples that follow.
  • a construct which is selectively expressed in the cells which are to be isolated.
  • a construct comprises a reporter gene which encodes a detectable gene product operably linked to a promoter which is specifically active in the cell type to be isolated.
  • suitable reporter genes include those encoding green, blue, yellow or red fluorescent proteins, ⁇ -galactosidase, ⁇ -glucuronidase, luciferase , or other proteins whose presence are visibly discernible in viable cells, of which green fluorescent protein is most preferred.
  • the reporter gene under the control of the cell type-specific promoter may encode a surface protein whose presence on the cell may be detected through the specific binding of an antibody or ligand.
  • the reporter gene under the control of the cell type- specific promoter may encode a protein that confers resistance to an antibiotic or other substance that induces cell death or apoptosis in cells lacking the expression of the protein.
  • the promoter selected be completely cell type specific.
  • a promoter expressed selectively, but not exclusively, in a cell type having a large size may be used in conjunction with a procedure that selects for larger cells.
  • Non- limiting examples of suitable promoters include, but are not limited to, the mec-3 and mec-18 promoters (specific for touch receptor cells), the unc-4, unc-30, unc-55, unc-25, unc-47 and ttx-1 promoters, all of which are nerve cell specific.
  • the construct may be incorporated into the intact organism, as set forth in the examples below, or may be incorporated after the organism is dissociated into individual cells using standard techniques.
  • organisms comprising the reporter construct are propagated to late stage embryos, which are then dissociated and sorted.
  • dissociated cells are then subjected to a sorting method which identifies the reporter gene product.
  • fluorescence-activated cell sorting methods may be used.
  • affinity-based methods known to those of ordinary skill in the art also may be employed to isolate the cells expressing the reporter gene encoding a protein expressed on the cell surface from those that lack expression of this same protein.
  • cell populations that specifically express a surface protein may be isolated using fluorescence-activated cell sorting if the antibody or ligand that specifically binds to the cell surface protein is conjugated to a fluorescent tag, a wide variety of which are known to the practitioner of ordinary skill in the art of cell sorting. Additional means of sorting, such as by size, viability or resistance to the otherwise toxic effects of certain exogenous substances, may also be performed.
  • the cells may be cultured prior to selection. In this way, substantial numbers of cells may be made available for sorting, providing a large source of RNA, an advantage over the prior art. Non-terminally differentiated cells may be further cultured after sorting.
  • the isolated cells may then be used to prepare cell type-specific RNA.
  • the resulting RNA may then be used directly as a probe, or may be amplified into RNA or DNA.
  • the nucleic acid to be used as a probe is detectably labeled.
  • Cell type-specific nucleic acid prepared as above, may then be used to screen a plurality of genes from C. elegans and/or other organisms.
  • arrays such as dot blot arrays or, more preferably, microchip-based nucleic acid arrays may be used.
  • control screening is performed using, as probe, nucleic acid generated from a reference population of C. elegans cells.
  • gene screening techniques well known to those of ordinary skill in the art may be useful in establishing the identity of novel genes present in the cell types isolated by the procedures described herein.
  • nucleic acids such techniques may include, but are not limited to SAGE (serial analysis of gene expression) or the screening of candidate genes by RT-PCR.
  • Protein chips may also be used to identify genes expressed specifically in the isolated cell populations either by simply applying to the chip a protein mixture obtained from the specific cell population isolated using the procedures described herein or by first translating in vitro the RNA isolated from the specific cell population and analyzing the proteins so obtained by means of mass spectroscopy, protein microarrays, or other techniques known to those of ordinary skill in the art that are suitable for protein identification.
  • Genes identified as being cell type-specific then may be analyzed to identify cell type-specific expression elements which then may be subjected to further study and use in model systems.
  • the present invention further provides wild-type and mutant nucleic acid and amino acid sequences for the C. elegans mec-5-dependent gene mec-17. Because mec-17 is important for maintenance of the differentiated state in C. elegans suppression of mec-17 gene expression by antisense or RNA interference methods known to those of ordinary skill in the art may be used for maintaining C. elegans cells in a non- differentiated state. Alternatively, forced expression of mec-17 through the introduction into target cells of vectors expressing the mec-17 gene may be used for triggering the terminal differentiation of C. elegans cells. Thus, the present invention provides methods for modulating the differentiation of C. elegans cells by modulation of mec-17 expression.
  • vectors useful for the introduction of nucleic acids that modulate cellular mec-17 gene expression include, but are not limited to, plasmids, cosmids, artificial chromosomes, or virus-based vectors known to be capable of introducing genetic material into C. elegans cells. Such vectors would be readily apparent to those of ordinary skill in the art.
  • RNA from whole animals with and without particular cell types like the six touch receptor neurons
  • DNA microarrays has an inherent sensitivity problem. Low abundance RNAs expressed in only a few cells or differences in RNA expression in particular cells are difficult to detect. Indeed, no touch cell genes were identified using RNA from wild- type and mec-3 mutant animals on DNA microarrays. This limitation was circumvented by obtaining and culturing wild-type and mec-3 mutant cells from embryos, isolating touch cells by cell sorting, and amplifying RNA from the isolated cells.
  • the isolated cells were identified by GFP fluorescence, expressed from the promoter for the touch cell-specific gene mec-18 for wild-type cells (the embryos have four touch cells: ALMLR and PLML/R) or the mec-3 promoter, which is expressed in the touch cells and two other neurons (FLPL/R) in the embryos (Way and Chalfie, 1989, Genes Dev. 3:1823-33), for mec-3 mutant cells. Both promoters are expressed after the touch cells are generated. The dissociated embryos yielded a very small number of faintly fluorescent cells. When cultured overnight, however, more cells (0.3-0.6%) elaborated processes and had intense GFP fluorescence.
  • GFP-positive cells which were presumably generated and/or differentiated in culture, were remarkably like in vivo touch receptor neurons in morphology (FIGURE 1) and gene expression (FIGURE 2; Table 1).
  • the wild-type cultured cells were usually monopolar with a single, long process or bipolar with a smaller second process 180° from the first, i.e., similar to the in vivo ALM and PLM cells, respectively.
  • the mec-3 cells were bipolar or multipolar with smaller processes that branched more randomly. This morphology is similar to that seen in vivo (Chalfie and Sulston, 1981, Dev Biol 82:358-70; Way and Chalfie, 1989, Genes Dev. 3:1823-33).
  • RNA from sorted wild-type cells and mec-3 cells was hybridized to genomic DNA microarrays containing DNA for 17,817 of the 18,967 known or predicted C elegans genes (Jiang et al, 2001, Proc Nail Acad Sci USA 98:218-23) to identify genes that were r ⁇ ec-3-dependent.
  • Three sets of independently prepared RNA samples from mutant and wild type touch cells were each hybridized onto two separate arrays. Seventy-one r ⁇ ec-3-dependent candidate genes were identified by choosing genes
  • genes could be either mec-3 repressed genes or genes that are expressed in a mec-3 -independent manner in the FLP cells. Gene expression was also compared in sorted mec-3 mutant cells and unsorted wild-type cells and identified 722 candidate genes that were enriched in the mec-3 mutant cells.
  • the arrays were spotted with coding-rich sequences from genomic DNA, and the DNAs for these three genes would not hybridize to at least 600 nt at the 3' ends of the RNAs.
  • the amplification methods employed preferentially produce sequences from the 3' ends of the RNAs.
  • DNA microarrays capable of detecting the 3' ends of mRNAs would have more accurately identified the mec-3-dependent genes.
  • RNAs from wild type and mutant cells All of these genes ranked below mec-3. As described below for the cct genes, genes giving small ratios can be differentially expressed in the touch cells. The modest difference in expression may result from more general gene expression or an indirect effect on gene expression resulting from the differentiation of the wild type cells. Altogether two of the 22 tested genes that were not previously known to depend on mec-3 had differential
  • MEC-3 maintains its own transcription and activates the mec-4 and mec-7 genes as part of a MEC-3 ::UNC-86 heterodimer (Xue et al, 1992, EMBO J 11:4969-79; Xue et al, 1993, Science 261:1324-8; Duggan et al, 1998, Development 125:4107-19).
  • the motif-finding program MEME (Bailey and Elkan, 1994, Proc Int Conflntell Syst Mol Biol 2:28-36) was used to define a consensus (FIGURE 3A) for these binding sites from these genes in C. elegans and two related nematodes C. briggsae and C. vulgaris.
  • oligonucleotide frequencies (Van Helden et al, 2000, Yeast 16:177-87) in the top twenty genes in Table 1 were also examined, and these studies identified a single heptanucleotide (AATGCAT) that was significantly overrepresented (P ⁇ 0.01). This motif matches the UNC-86 binding core in the consensus sequence (FIGURE 3A), and, thus provides a proof-of-principle that such binding sites can be deduced from the array data. Twenty-eight of the 71 genes in Table 1 have this heptanucleotide. Consistent with the hypothesis that genes showing strong enrichment are direct targets of mec-3 regulation, 13 of the 16 genes with expression ratios from real time RT-PCR
  • the genes reflect the general distribution of genes on all six chromosomes. Most mec-3-dependent genes are found in the gene clusters on the autosorn.es, but some are also found near the ends of the chromosomes. The genes are also fairly uniformly distributed on the X chromosome, which has no cluster. In general, the six touch receptors do not use genes from specific parts of the chromosomes.
  • Table 1 contains unc-24, whose product shares a stomatin-like region with MEC-2 (Barnes et al, 1996, J Neurochem 67:46-57) and acr-13, an acetylcholine receptor subunit (two other such subunits are expressed in the touch cells (Treinin and Chalfie, 1995, Neuron 14:871-7; Treinin et al, 1998, Proc Natl Acad Sci USA 95: 15492-5) but are not included in Table 1).
  • Table 1 may well contain genes providing functional redundancy, but redundancy through the use of multiple members of gene families does not seem to play a numerically important role in touch cell differentiation.
  • the touch receptor neurons have a distinctive morphology and are not found as yet in non-nematode species, the genes used by these cells are not more nematode-specific than those of the entire C. elegans genome. Thirty-eight percent (27/71) of the genes in Table 1 are similar to genes in other organisms, compared with 36% for all C. elegans genes (Rubin et al, 2000, Science 287:2204-15).
  • Table 1 provides a large number of genes to test for their effects on the development and function of the touch cells.
  • the highest-ranking gene in Table 1, F57H12.7 is an example of such a gene.
  • F57H12.7 maps to the same position of the previously uncloned mec-17 gene, mec-17 appears to be needed for the maintained differentiation of the touch cells; mec-17 mutants are touch sensitive at hatching, but become insensitive as they mature (Chalfie and Au, 1989, Science 243:1027-33 (1989). Expression of mec-3 and mec-7 is reduced in older mec-17 animals (Way and Chalfie, 1989, Genes Dev.
  • F57H12.7 was identified as mec-17 because the single mutant allele of mec-17, u265, contained two missense mutations (FIGURE 4A) and microinjection of F57H12.7 genomic DNA into mec-17 (u265) animals rescued the mutant phenotype.
  • F57H12.7 is expressed solely in the touch cells; as the GFP fusion protein was very strongly expressed in these cells from late embryos to adults (FIGURE 4B). This expression depends on mec-3, since no fluorescence was seen when the fusion was expressed in mec-3 mutants.
  • mec-17 encodes a predicted polypeptide of 262 amino acids. Because the protein is novel with no readily identifiable motifs, the sequence does not provide any hints as to how mec-17 may act to maintain the expression of mec-3 and the differentiation of the touch cells. Nonetheless, mec-17, the one other predicted C. elegans gene, W06B11.1, and two predicted genes each from Drosophila, mouse and humans share a 158 amino acid domain with 29-41% identity (FIGURE 4A). This shared domain suggests that these proteins may function similarly.
  • genes in Table 1 especially those with strong mec-3- dependent expression present interesting questions about their roles in the function of these cells.
  • Such genes include twk-28 (encoding a TASK potassium channel protein), ZC482.5 (GABA receptor subunit), four genes encoding predicted enzymes or proteins with catalytic domains [C03A3.3 (metallo- ⁇ -lactamase), F14E5.4 (acid phosphatase), and F20D6.5 (tyrosine kinase), and F01D5.8 (esterase)], two genes (C06A8.3 and far-3) encoding proteins similar to antigens from the parasitic nematode Onchocerca volvulus, and four genes (rpl-20, rps-18, rps-18 and rps-14) encoding ribosomal proteins.
  • the last set of genes, the cct genes, and perhaps others may be enriched in the wild-type touch cells as a secondary consequence of the activation of other genes (e.
  • RNA from whole animals has been applied to DNA microarrays to see gross differences in expression between the male and hermaphrodite animals (Jiang et al, 2001, Proc Natl Acad Sci USA 98:218-23) or germ lines (Reinke et al, 2000, Mol Cell 6:605-16), which make up approximately two thirds of the cells in adults, or between animals at different developmental stages (Hill et al, 2000, Science 290:809-12; Jiang et al, 2001, Proc Natl Acad Sci USA 98:218-23).
  • DNA microarrays can be used to identify potential differentiation targets for single types of cells in multicellular organisms and these techniques should be applicable toward the study of cellular development and function under a variety of experimental conditions.
  • RNA from the sorted cells was amplified through two rounds by a modification of the method of Eberwine et al. (Eberwine et al, Proc Natl Acad Sci USA 89:3010-4). Real time RT-PCR. Approximately 3-5 ⁇ g of second round amplified RNA from either purified wild type or mec-3 (el 338) touch cells was converted to cDNA using random primers and PowerScript reverse transcriptase (Clontech) according to the manufacturer's protocol.
  • RNA samples were generated from 3 independent primary cell cultures and cell sortings for wild-type touch cells (wl, w2 and w3) and for mec-3(el338) touch cells (ml, m2 and m3).
  • Each hybridization (wl-ml, w2-m2, w3-m3) was done twice. All non-flagged spots for which the net fluorescence intensity at least in one channel was three background standard deviations above background were considered well hybridized and measured. If the net fluorescence intensity for a non-flagged spot was within one standard deviation of the background, net fluorescence intensity was set as one standard deviation of the background. The natural logs of the ratios for the duplicate hybridizations were averaged. T-test P values were determined with the averages from three independent experiments.
  • the motif-finding program MEME (Bailey and Elkan, 1994, Proc Int Conf Intell Syst Mol Biol 2:28-36; meme.sdsc.edu/meme/website/meme.html) was used to derive a 19 nucleotide consensus sequence from 19 known MEC-3 ::UNC-86 binding sites in the promoter region of mec-3, mec-4 and mec-7 in C.elegans, C. briggsae, and C. vulgaris.
  • a weight matrix for the defined consensus site was built based on the multiple sequence alignment and was used to search the 5' UTRs of the genes in Table 1 for the significant matches (P ⁇ 10 "4 ) using the pattern search tool at www.ucmb.ulb.ac.be/bioinformatics/rsa-tools/.
  • a graphical representation of the weight matrix was generated using the tool available at ep.ebi.ac.uk/EP/SEQLOGO/.
  • Protein-protein blast (BlastP) (www.ncbi.nlm.nih. gov/BLAST with the non- redundant protein data base at NCBI and with C. elegans proteins at Wormbase ( " www.wormbase.org) was used to identify similar genes, i.e. those with an expectation value (E) ⁇ 10 ⁇ 10 and that are aligned through at least 80% of their lengths as in Chervitz et al. (Chervitz et al, 1998, Science 282:2022-8).
  • the mec-17(u265) genomic fragment was amplified by PCR from worm lysates.
  • the PCR product was sequenced by GeneWiz Inc., New York.
  • a 5.5 kb fragment of mec- 17 genomic DNA was amplified by PCR using the Clontech polymerase mixture and
  • the plasmid (0.75 ng/ ⁇ l) was injected with the rol-6(sul006) marker plasmid ⁇ RF4 (Mello et al, 1991, EMBO J
  • mec-17::gfp was generated by inserting about 5 kb genomic DNA fragment (produced by PCR) including 1.9 kb upstream sequence and all the predicted genomic sequence except for the 42 amino acids at the C-terminus of MEC-17 into pPD95.77 (a gift from A. Fire), which contains the gfp gene with added introns.
  • the construct was transformed with DA#735 (Huang et al, 1994, Mol Biol Cell 5:395-411), a plasmid containing lin-15(+) DNA, into MT1642 [Un-15(n765J ⁇ .
  • Transgenic worms from three stable lines were examined using an Axiophot microscope with an FITC filter set.
  • the cct-4::gfp construct was generated by inserting 2389 bases of cct-4 genomic DNA including 585 bases of upstream sequence in frame into pPD95.77.
  • PcctX 'gfp was generated by inserting 2436 bases of upstream sequence of cct-2 and 42 base of coding sequence in frame into pPD95.77.
  • the construct and pMH86 were injected into TU353.
  • Hermaphrodites with extrachromosomal arrays from three stable lines were mated with dpy-20(el282) males to obtain touch sensitive mec-3/+ heterozygotes that were examined for fluorescence.
  • Y48G8AL.8 Contains ribosomal protein L22 signature 3.13 0.032 cct-1 T-complex chaperonin protein 2.10 0.002 4 mec-3 LI homeodomain transcription factor 3.09 0.014 7 C02F5.3 GTP-binding protein 2.10 0.047 rpl-18 Ribosomal protein L18 3.05 0.047 R05D7.5 Unknown 2.09 0.038
  • C Isolation of cells from the embryos. Divide the staged embryos between two 50 ml conical tubes, wash embryos IX with 25 ml egg buffer (D3), and remove as much of the buffer as possible. Resuspend the embryos in 2 volumes (usually 20 ml per tube) of chitinase/chymotrypsm solution (D4) and gently shake at room temperature. Monitor the embryos by DIG to determine that the eggshells have been digested (usually 5 min - extended treatment damages the cells). Wash the embryos with egg buffer and pellet them at 2,000 rpm for 1 min.
  • D3 chitinase/chymotrypsm solution
  • Concentrated OP50 Culture OP50 (Brenner, 1994, Genetics 77:71-94) overnight at 37°C in 12 L of Superbroth (Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), pellet, and resuspend in 60 ml of M9 buffer (final volume approximately 120 ml). Store at 4°C until needed.
  • Hypochlorite solution 1 ml sodium hypochlorite (Sigma), 2.5 ml 1 M NaOH, 1.5 ml dH 2 O.
  • Egg buffer 11.8 ml 1 M NaCl, 4.8 ml 1 M KC1, 0.34 ml 1 M CaCl 2 , 0.34 ml 1 M MgCl 2 , 2.0 ml 0.25 M HEPES (pH 7.4), 82.5 ml dH 2 O. Filter-sterilize and store at
  • Chitinase/chymotrypsin 5 mg chitinase (Sigma C-6137), 5 mg alpha- chymotrypsin (ICN 152272), 1 ml egg buffer, 10 ⁇ l penicillin-streptomycin [Cat. No. 15140-148, 10,000 units of Penicillin (base) and 10,000 ⁇ g of Streptomycin base/ml, Gibco] . Freeze in 10 ml aliquots at -20°C.
  • L-15CM 24 ml L-15 (Gibco), 6 ml inulin [ICN 102055; 7.5 mg/ml egg salts (D7), autoclaved and stored at 4°C], 0.4 ml base mix (D8), 200 mg PVP (polyvinylpyrrolidone, Sigma), 0.4 ml penicillin-streptomycin (Gibco). Filter- sterilize, add 8 ml fetal bovine serum (Heat-inactivated, Sigma), and store at 4°C until needed on the same day.
  • L-15SFM 24 ml L-15, 6 ml inulin, 0.4 ml base mix, 10 ml egg buffer, 0.4 ml penicillin-streptomycin, 100 mg PNP. Filter-sterilize and store at 4°C. 7.
  • TESPA-coated slides Soak microscopic slides in 70% ethanol and 1% HI for 1 to 2 hours. Rinse 3X with dH2o Air-dry and sterilize slides under a culture hood UN lamp for 1 hr (all subsequent steps should be performed under sterile conditions). Soak slide in 4% TESPA (3-aminopropyltriethoxysilane, Sigma)/acetone solution for 5 min. Wash slides in acetone twice. Air dry. Soak slides in 4% paraformaldhyde/PBS solution for 30 min at room temperature. Wash slides in sterile water for four times. Air dry and store at room temperature. Treated slides have to be used within 3 days.
  • Base mix 100 ml H 2 O, 100 mg adenine, 10 mg ATP, 3 mg guanine, 3 mg hypoxanthine, 3 mg thymine, 3 mg xanthine, 3 mg uridine, 5 mg ribose, 5 mg deoxyribose; autoclave and store at 4°C.
  • Enrichment cell sorting used a fluorescence threshold trigger (FLTT) instead of the standard light scatter threshold trigger (LSTT) (McCoy et al, 1991, Cytometry 12:268-74). This technique helps reduce the analysis of small particles and negative cells and allows for higher throughput of cell suspensions from disassociated embryos.
  • FLTT fluorescence threshold trigger
  • LSTT standard light scatter threshold trigger
  • RNA quality decreases, however, with the extended storage.
  • 8 ⁇ l of the reaction can be mixed in another tube with 0.8 ⁇ l ⁇ -P 32 -CTP. Incubate both tubes at 37°C for 18 hours. Purify transcripts from cold reaction using the RNeasy Minikit. Precipitate the hot reaction with 5% trichloroacetic acid and 20 niM sodium pyrophosphate to determine the amount of transcription (Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 2 n edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • 5X second stranded buffer 500 mM KCl, 50 mM (NH 4 ) 2 SO 4 , 25 mM MgCl 2, 0.75 mM NAD + (nicotinamide adenine dinucleotide), 100 mM Tris-Cl (pH 7.5), and 0.25 mg/ml BSA.
  • 10X KFI buffer 200 mM Tris (pH 7.5), 100 mM MgCl 2 , 50mM NaCl.

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Abstract

L'invention concerne des procédés visant à isoler des types spécifiques de cellules du nématode Caenorhabditis elegans, à isoler de l'ARN de celles-ci et à amplifier l'ARN collecté afin de produire de l'acide nucléique amplifié, à exposer l'acide nucléique amplifié, dans des conditions strictes d'hybridation, à une collection d'acides nucléiques de référence représentatifs d'une pluralité de gènes de nématode, et à identifier les acides nucléiques de référence particuliers qui s'hybrident. Les acides nucléiques de référence qui s'hybrident le plus fortement, en particulier ceux qui présentent une hybridation supérieure par rapport à des taux d'hybridation témoins obtenus à l'aide d'acide nucléique issu d'autres cellules de C. elegans ou d'une population générale de C. elegans, peuvent représenter des gènes exprimés de manière sélective dans le type spécifique de cellule isolée. Les séquences d'acides nucléiques des régions flanquantes des gènes spécifiques dudit type de cellule peuvent être analysées en vue d'identifier des éléments d'expression spécifique de ce type de cellule.
PCT/US2003/001632 2002-01-18 2003-01-17 Procedes d'isolement de cellules specifiques de caenorhabditis elegans et d'obtention d'informations genomiques a partir de celles-ci WO2003062449A2 (fr)

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