Patent Application: US-93241301-A

Abstract:
a method of using elongate multicellular organisms in conjunction with a specialized flow cytometer for drug discovery and compound screening . a stable , optically detectable linear marker pattern on each organism is used to construct a longitudinal map of each organism as it passes through the analysis region of the flow cytometer . this pattern is used to limit complex data analysis to particular regions of each organism thereby simplifying and speeding analysis . the longitudinal marker pattern can be used to alter signal detection modes at known regions of the organism to enhance sensitivity and overall detection effectiveness . a repeating pattern can also be used to add a synchronous element to data analysis . the marker patterns are established using known methods of molecular biology to express various indicator molecules . inherent features of the organism can be rendered detectable to serve as marker patterns .

Description:
the following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention . various modifications , however , will remain readily apparent to those skilled in the art , since the general principles of the present invention have been defined herein specifically to provide improved data processing of optical signals from elongate multicellular organisms by use of a pattern of markers of spaced apart along the long axis of the organisms . a general approach to creating a marker strain of organisms is to genetically introduce a set of features that are readily detected by a flow cytometer . a simple approach is to produce features that can be directly detected by their fluorescence - for example by introducing a gene for a fluorescent protein . any detectable pattern can be used , however . enzyme patterns can be detected by histochemical reactions producing a colored or fluorescent product . proteins can be overexpressed so as to be optically detectable . other biological products such as fat globules , crystals or natural pigments can also serve to form an optically detectable pattern . the pattern could be antigenic and be detected by of antibodies , or the pattern could be carbohydrate - based and detectable by addition of lectins . the lectins and antibodies can be fluorescent , or can be linked to histochemically detectable molecules or optically detectable structures such as microspheres . although in most instances it will be necessary to employ genetic manipulation to produce an optimal marker strain , some naturally occurring organisms or strains of organisms have cryptic marker features that can be revealed through the application or antibodies , histochemicals or other such methods . in the case of genetic manipulation it is advantageous to select a promoter that will result in a desired spatial pattern of expression . an example of such a promoter is the egl - 17 promoter of c . elegans . this promoter sequence , when inserted at the 5 ′ end of a gene , will result in expression of the gene product ( protein ) in the m4 neuron and in vulval precursor cells of the organism . the positions of these cells are well characterized and are invariant in a wild - type ( n2 ) background . the gene controlled by the chosen promoter should encode a detectable product . an example of such a gene product is a fluorescent protein such as the asred gene ( clontech , inc .). as already mentioned , a large variety of other detection methods are available such as those involving enzymatic or antigenic properties . an advantage of a fluorescent protein is that the organism can be analyzed directly with no need for special incubations or other sample preparation . standard molecular genetic techniques are used to clone the promoter dna sequence , the detectable protein gene sequence , and other dna sequences required for optimal expression in the organism into an appropriate plasmid vector . for example , the present inventors and their associates have constructed a series of expression vectors in which a synthetic intron has been inserted at the 5 ′ end and the c . elegans unc - 54 , a 3 ′ utr ( untranslated region ), has been inserted at the 3 ′ end of each clontech reef coral protein gene ( amcyan , zsgreen , zsyellow , dsred , dsred2 , or asred ). the egl - 17 promoter sequence has been inserted upstream of the 5 ′ synthetic intron in each of the expression vectors resulting in egl - 17 expression plasmid constructs for each fluorescent protein . the expression plasmid dna is then inserted into the genome of the host organism . one method used for c . elegans entails microinjecting plasmid dna into the gonad of young adult hermaphrodites and selecting progeny that express the detectable marker . such animals generally carry the marker dna as an unstable extrachromosomal array . additional steps are required to cause the dna to become integrated into a chromosome and to select the progeny bearing this integration . this is generally accomplished by mutagenizing the animals to introduce random double stranded breaks in chromosomal dna . during the dna repair process extrachromosomal sequences can become incorporated into a chromosome . f 2 progeny that have undergone such an incorporation event can be screened . f 2 homozygotes from such an integration event are identified based on their ability to transfer the marker dna to 100 percent of their progeny . it should be noted that other methods , including some that result in integration into a specific site in a chromosome can also be used . the point of the present invention is use of the pattern - marked organism as opposed to creation of such an organism . [ 0029 ] fig1 shows photomicrographs of transgenic organisms where expression of a fluorescent protein is under the control of the egl - 17 promoter . in this case the construct is egl - 17 :: zsyellow . fig1 a and 1b show light micrographs of two organisms with fig1 c and 1d showing the corresponding fluorescence images with the head ( h ) and vulva ( v ) marked . diffuse autofluorescence of the gut is discernible between the head and vulva . fig2 shows oscilloscope traces of the optical detector signal from flow cytometric analysis of these organisms . fig2 a and 2b show oscilloscope traces from representative egl - 17 :: zs yellow expressing c . elegans . an extinction signal 30 indicates when the organism enters and exits the laser beam . in the above - referenced co - pending application it is disclosed that forward light scatter measured over a wide solid angle is often a more effective discriminator than simple extinction . it should be understood that all references herein to extinction can be replaced by wide - angle forward light scatter or another signal shown to effectively indicate presence of an organism . the upper trace 60 represents yellow fluorescence . the yellow fluorescence signal 60 is indicative of the presence of zsyellow and marks the head and vulva in the organism . these precisely located points of fluorescence represent a marker pattern as used in the present invention . these results should be compared with the trace ( fig2 c ) of a control organism lacking the genetic construct . in cases where mutagenesis has been employed , it is advantageous to remove extraneous mutations by performing several rounds of mating with wild - type organisms and selecting for homozygotes for the inserted marker . next , the marker must be transferred to an appropriate background strain for the planned assay by mating . for example , in a ras pathway assay for new pharmaceuticals one could perform the screen using a c . elegans lin - 15 mutant that already contains a second or possibly a third detectable marker . in that case the positional marker pattern generated above would be transferred by mating into the lin - 15 strain . if only fluorescent markers were going to be utilized , one can simply mix the different dnas prior to insertion into the genome thus simultaneously adding all markers into the appropriate strain . the desired background is one that shows an optically detectable response to an active compound . this allows the organisms to be used to screen compound libraries for drug candidates . the marker pattern ensures that the detected signal is positionally correct for the screened activity . that is , it is quite likely that test compounds may have multiple activities that could result in positional changes in the expressed signal and / or anomalous expression . the marker pattern allows the system to discriminate between positional shifts in expression . as explained below , the pattern is especially effective in enabling detection of weak signals resulting from test compounds . using marker patterns to detect suppression of a disease model phenotype certain disease model pathways involve the inappropriate activation of gene expression in certain tissues or in the migration of certain cell types during development of the animal ( which then results in positional changes in marker expression ). one such model involves the wnt signaling pathway in c . elegans . components of this pathway appear to be conserved in other organisms and have been shown to function in the development of several forms of cancer , including breast cancer ( nusse and varmus , 1982 ; lejeune et al ., 1995 ) and colon cancer ( morin et al ., 1997 ; rubinfeld et al ., 1997 ). wnt signaling in c . elegans is involved in controlling the migration of specific cells ( korswagen et al ., 2000 ). one example is that the proper migration of the ql neuroblast descendants depends upon the proper expression and function of the wnt pathway genes mab - 5 and egl - 20 ( kenyon , 1986 ; salser and kenyon , 1992 ; harris , et al ., 1996 ). if the ql neuroblasts are marked with a fluorescent protein gene , the distance of these cells , which are normally located in the tail of the animal , from the vulva can be measured using marker pattern organisms . note that the distance between the vulva signal and the m4 neuron signal in the pharynx allows for precise correction for the size of the animal and thus acts as an internal control . the disease model mutant displays inappropriate migration of these cells , or inappropriate expression of fluorescent protein in other cell types . in such a case , a high throughput drug discovery assay involves exposing the animals to compounds and determining which compounds caused the animals to assume a wild - type fluorescence pattern . the marker pattern enables the analysis to readily detect the shift of the positionally incorrect signal into a normal wild - type position . by allowing the signal analysis to focus on limited regions of the organism and / or by allowing a switching of the analysis mode ( e . g ., peak detection to integration ) within specific ( lineal ) regions of the organism , the invention also allows the unambiguous detection of weak fluorescence signals . in some cases the autofluorescence ( intrinsic fluorescence of the organism ) signal of an organism is great enough to obscure the signal of a marker . in the case of c . elegans py1089 gfp ( green fluorescent protein from aequorea victoria ) is expressed in two adjacent neurons in the head of the animal . that fluorescence is visible under the microscope as two areas of more concentrated green fluorescence in a background of diffuse autofluorescence . current automated analytical instrumentation integrate the total fluorescence signal of the organism and are therefore not sensitive to the brighter region within the autofluorescence . attempts to resolve this strain from wild type c . elegans using such automated systems have been unsuccessful thus far . when the electronic signal from the organism is monitored a clear peak signal is seen at one end of the animal . by synchronizing the signal analysis to a known marker pattern , it is possible to determine the orientation of the animal ( e . g ., head first ) and analyze only the specific gfp signal from the head neurons . in the case of the animal whose oscilloscope traces are depicted in fig3 d , the total area under the fluorescence profile is 550 units while the area under the fluorescence peak is only 50 units resulting in a signal to noise ratio of 1 : 10 . if , however , the area under the fluorescence peak is compared to an area of comparable width in the region of the animal with the highest autofluorescence that ratio changes to 2 : 1 . if one then considers that the fluorescence signal from the two nerve cell bodies is 35 units , an assay is able to detect the presence of a third fluorescent cell body or the loss of one of the two fluorescent cell bodies . a useful marker in this situation is the egl - 17 positional markers described above . with egl - 17 :: zsyellow as the positional marker the instrument detects the m4 neuron in the anterior portion of the pharynx and the vulval precursor cells and rapidly determines the orientation of the animal as it passes through the analytical chamber . the software looks for the first green fluorescent peak immediately posterior to the m4 neuron and displays the intensity of only that signal . results include signals such as 18 ( no gfp fluorescence ), 35 ( gfp fluorescence in only one cell ), 50 ( fluorescence in two cells ), 68 ( 3 cells ), and 86 ( 4 cells ). [ 0037 ] fig3 shows light and fluorescence photomicrographs of a c . elegans py1089 animal are shown in fig3 a and fig3 b , respectively . oscilloscope traces depicting the optical detector signals generated by two different py1089 animals are shown in fig3 c and fig3 d . in fig3 b the fluorescence from the head neurons ( lower end of imaged organism ) is clearly visible and is distinguishable from the overall autofluorescence of the animal . here the animal is oriented such that the two neurons are aligned one on top of the other and only one slightly diffuse spot is observed . in fig3 c and 3d the one of the traces 30 shows the extinction signals from the animals while the other trace 50 show the green fluorescence signal . the animal in fig3 d was longer than the animal in fig3 c and was probably an adult . as expected , both the peak autofluorescence and the total autofluorescence ( the area under the curve ) are larger for the larger animal . the total fluorescence from the head neurons is approximately the same for both animals . for fig3 d the total area under the fluorescence curve is 542 units while the area under the gfp peak is 53 . of the peak area approximately 11 . 5 units are due to autofluorescence while the remaining 41 . 5 are due to the gfp fluorescence . these measurements indicate that discrimination could be made automatically provided that a marker pattern is available to automate decisions concerning the organism &# 39 ; s orientation . [ 0038 ] fig4 is a graphic representation ( oscilloscope ) of the optical detector signals from an egl - 17 :: zs yellow construct mated into py1089 . the presence of an organism is determined by an extinction signal 30 . a yellow fluorescence signal 60 ( zsyellow ) clearly marks the head end of the organism ( sharp spike used by the software to determine orientation ) and provides several other fluorescence peaks along the length of the organism . a more diffuse green fluorescence signal 40 ( gfp ) is then integrated to determine the head neurons , which immediately follow the yellow m4 neuron peak . the various marker patterns provided by the present invention allow the software to determine the orientation of elongate organisms , allow the software to specifically measure the position of treatment dependent signals ( by comparison to invariant marker pattern signals ), allow the software to alter the mode of signal analysis ( e . g . peak detection versus signal integration ) in a positionally controlled manner , and allow the software to limit detailed data analysis to specific positions along the length of the test organism . from the forgoing description a number of uses of the marker pattern organisms will be apparent to those of skill in the art . one method is to produce a test organism that expresses a marker pattern and also variably displays a detectable signal in response to one or more treatments . generally a treatment will be exposure of the test organism to one or more test compounds , for example , to select active drug candidates from a synthesis library . however , the treatment may also include one or more environmental or other factors that potentiate or otherwise affect the action of the test compound . after the exposure to the treatment , the test organism is analyzed by a flow cytometer . the marker pattern is detected and the analytic software of the system uses the marker pattern to effectively analyze the signal that represents treatment response . as explained above , such analysis would be impossible or much less efficient without use of the marker pattern . it will be appreciated that a major goal is to select out organisms on the basis of their response to the treatment . this requires that data analysis be completed before the organism passes through the sorting section of the flow cytometer . therefore , data analysis time is very brief and the enhanced analysis permitted by the use of marker patterns is often crucial . the following claims are thus to be understood to include what is specifically illustrated and described above , what is conceptually equivalent , what can be obviously substituted and also what incorporates the essential idea of the invention . those skilled in the art will appreciate that various adaptations and modifications of the just - described preferred embodiment can be configured without departing from the scope of the invention . the illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention . therefore , it is to be understood that , within the scope of the appended claims , the invention may be practiced other than as specifically described herein . burdine , et al ., “ egl - 17 encodes an invertebrate fibroblast growth factor family member required specifically for sex myoblast migration in caenorhabditis elegans ,” proc . natl . acad . sci ., u . s . a . 94 : 2433 - 2437 . burdine , et al ., “ egl - 17 ( fgf ) expression coordinates the attraction of the migrating sex myoblasts with vulval induction in c . elegans ,” development 125 : 1083 - 1093 . byerly , l ., r c . cassada and r l . russell . machine for rapidly counting and measuring the size of small nematodes . rev sci instrum . 1975 may ; 46 ( 5 ): 517 - 22 . harris , j ., honigberg , l ., robinson , n ., and kenyon , c . 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