Lexicon Phenotypic Analysis User Manual
Contents
1. CATScan images movies DEXA graphs data MicroCT images graphs data X ray images Neurology Therapeutic Section Data may be available for the following Assays in this section Basal Body Temperature graphs data Circadian Rhythms graphs data Functional Operational Battery data Formalin Assay graphs data Hot Plate graphs data Inverted Screen Assay data Open Field Test graphs data Repulse Inhibition Assay graphs data Rotarod graphs data Stress Induced Hyperthermia graphs data Tail Suspension graphs data Trace Aversive Conditioning graphs data Trace Conditioning graphs data Oncology Therapeutic Section Data may be available for the following Assays in this section e Skin Proliferation Assay graphs data i Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Ophthalmology Therapeutic Section Data may be available for the following Assays in this section e Fundus Photography images graphs data e Angiogram Photography images Pathology Therapeutic Section Data may be available for the following Assays in this section e Gross Observations data e Organ Tissue Weight data e Microscopic Analysis pictures data Minimum System Requirements e For Microsoft Windows An Intel Pentium 133Mhz or equivalent processor running Windows 95 or higher including Windows 2000 or2. 33 19 53 047 F 119 het 106 RW 0 0712 0 0293 3 5 25 35 63 064 F 125 het 116 R FL O 0f66 0 029 0 5 24 61 17 26 O46 F 125 het 116 FR FR 0 0718 0 0291 0 0 24 61 15 02 O44 F 125 het 119 RW 0 0714 0 0366 D o 24 61 6 42 1 08 F 125 het 119 T Animal Total 0 0537 0 5405 0 0 24 61 14 24 2363 h 1 31 het 103 T Animal Total 0 0504 0 5334 10 1 30 69 14 95 20 00 hi 131 het 103 FW 0 09 00388 10 1 230 69 109 O79 h 4 31 het 103 R FL O 065 0 0319 10 1 20 69 16 935 0 69 bl 131 het 103 FR FR 0 0833 0 029 10 1 30 69 1646 0 51 hM 1 39 het 119 T Animal Total 0 057 0 5654 10 0 36 39 25 09 34 75 M 139 het 116 FW 0 065 0 0303 10 0 S659 12 59 1 07 h 1 9 119 R FL 0 1041 0 0389 10 0 36 39 29 29 0 56 M 1 39 119 R FR 0 0913 0 0362 10 0 S659 22 19 053 F 59 ee T Animal Total 0 0501 0 4954 9 6 2616 209 2453 F 59 112 RW Of 0 029 ego Seka Gode Figure 13 DEXA Raw Data Table Note that by clicking on the spreadsheet icon when viewing a raw data table you can export and open all available raw data directly in Microsoft Excel or save it to disk Other Available Data Types Data is also available as images depending on the Assay Clicking on image thumbnails will often provide a specific image along with an annotated example The figure below shows a fundus photography picture and its annotated control picture Image Comparator Mouse Label F 125 Comments None Reference Image Figure 14 Fundus Photography Picture amp Annotated Control f Lexicon Copyright 200
3. This type of graph bar graph is used to show the proportion of animals that show either a Normal filled color or Abnormal unfilled color test result In this example Urinalysis is performed to test the level of Nitrites in the Urine for all animals In this case 3 out of 3 wild type animals 1 out of 1 heterozygous animal and 3 out of 5 homozygous animals show normal Nitrite values and 2 out of 5 homozygous animals show abnormal Nitrite values Such a representation is useful to report on the proportion of animals in each distribution whose results match a specific condition Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Line Graphs OPEN FIELD Center to Total Dist Ratio mar ot EST RSiwth 4 4 414 RSiheti 474 414 RSthom E E 6 8 1 00 vi Ci ni o k Mie sa wi Ci i a Ll T rit He 414 414 414 4 4 B B 8 8 4 Minute Intervals Figure 6 Line Graphs Lexicon Lexicon Phenotypic Analysis Software Documentation This figure shows a typical line graph where datapoints were collected over a period of time Such displays can be useful in addition to a dot plot graph for each interval to show a trend in the data Habituation or learning can be an important factor to consider when assessing the existence of a behavioral phenotype The top of the figure shows the number of datapoints that are represented for each interval measurement and with which
4. Trapping Null Allele Point Mutations Conditional etc In such cases there are several Lexicon Phenotypic Analysis Projects available for each type of mutation generating potentially different alleles within the same gene Throughout these pages a Lexicon Phenotypic Analysis Project is assigned a unique identifier consisting of a LEXKO prefix and a 3 or 4 digit number ex LEXKO 443 Gene Mutation Strategies Gene Targeting by Homologous Recombination Gene Targeting by Homologous Recombination affords deliberate and precise manipulation of genomic sequence in the mouse and is the process used to replace a specific gene sequence with a mutant exogenous sequence such as a positive selection cassette and marker gene When this procedure is performed in mouse embryonic stem ES cells mice with the corresponding mutation can be produced A typical targeting vector contains a selectable marker flanked by regions of homology to the gene of interest and is introduced into ES cells by electroporation Once in the cell the vector aligns with the corresponding genomic sequence and the corresponding region of the gene is replaced by the selectable marker creating the desired mutation through an homologous recombination event ES cells that have incorporated the targeting vector can be enriched under selection The surviving stem cell clones are then subjected to further characterization to ensure that the desired homologous recombination ev
5. Validator Software The Lexicon Mouse Production Software is primarily a workflow system used to manage the generation and breeding of Knockout mice It is responsible for producing gathering and recording all relevant mouse and colony information and tracks all mice by allele through each phase of a project lifecycle The Lexicon Mouse Production Software generates numerous reports calendars and queues for managing the process of producing all required mice for later phenotypic analysis It is also used to ensure that all participants in colony management know when an action needs to occur and when data is ready to be collected for a particular set of mice using the Lexicon Phenotypic Data Collector Software The Lexicon Phenotypic Data Collector Software is used to upload physiological or medical data collected for each of the analyzed mice bred and genotyped through the Lexicon Animal Production Software The Lexicon Phenotypic Data Validator Software is used to perform various data validation checks and guide the manual review of phenotypic data before it is verified and migrated to the Lexicon Phenotypic Analysis Database where it can be analyzed and reviewed by research scientists This software also provides tools for annotating and summarizing the phenotypic observations of mouse gene knockouts compared to wild type controls and historical background data The Lexicon Phenotypic Analysis Software is used by research scientists to assess t
6. distributions from homozygous or heterozygous animals to their wild type littermates In addition comparisons can be made to the distribution of all historical wild type values for a particular assay as described later in this document Therapeutic Section In order to classify experiments that are performed on mice the data from particular Assays are organized into different Therapeutic Sections Each of these sections will contain a list of specific Assays so that results can be easily and consistently retrieved throughout the software for each Lexicon Phenotypic Analysis Project The software comes pre configured with the following standard Therapeutic Sections Blood Chemistry Tests Cardiology Assays Diagnostics Assays Gene Expression Data Immunology Assays Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Neurology Assays Oncology Assays Ophthalmology Assays Pathology Reports Metabolism Assays Assays Data collected for a particular project are organized by Assay An Assay is interpreted as a means of collecting data from a particular instrument or through a web form that a researcher has populated through Lexicon Phenotypic Data Collector Software The Lexicon Phenotypic Analysis Software currently supports the collection and visualization of data from more than 70 different analytical procedures or instruments Displays of gender specif
7. such as 1 Gene classification by functional category and cell location 2 Description of the mouse gene as well as Known human orthologs 3 Short summary of known or putative biology of the target with relevant literature and database citations when available Phenotypic Summary Phenotypic Summary LEAKO 011 The homozygous mutant mice exhibited increased peripheral blood percentages of CD4 cells and decreased B cells when compared with their wild type littermates and the historical means Decreased lgG1 response to ovalbumin was noted in mice In addition the male mutants exhibited signs of growth retardation including decreased mean body weight and length total tissue mass and lean body mass RT PCR analysis revealed that the transcript was absent in the homozygous mutant mice This section gives an overall description of the relevant phenotypic observed for the gene specific mutation f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Mouse Gene Sequence Information Mouse Gene Sequence Information Nucleotide NM 028817 ACCESSION NM_028817 NID gi 75992919 ref NM_028817 2 Mus musculus acy Co Reference synthetase long chain family member 3 Acsl3 transcript variant 1 mRNA Protein Reference Entrez Gene 14205 USCAW4 OSC4W4 09CZW4 2610510B12RIK PROTEIN Figure 19 Mouse Gene Sequence Information This secti
8. 028817 NID gi 75992919 ref NM_028817 2 Mus musculus acyl CoA Reference synthetase long chain family member 3 Acsi3 transcript variant 1 mRNA Protein Q9CZW4 Q9CZW4 QSCZW4 2610510B12RIK PROTEIN Reference Entrez Gene 74205 Human Gene Sequence Information 3 Nucleotide NM_004457 ACCESSION NM_004457 NID na Homo sapiens Homo sapiens fatty acid Coenzyme A Reference ligase long chain 3 FACL3 mRNA hs_refseq Protein 095573 LCFC_HUMAN 095573 LONG CHAIN FATTY ACID COA LIGASE 3 EC Reference ae Entrez Gene 2181 Mutation Information Figure 17 Gene Summary Page Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Gene Summary Gene Summary for LEXKO 011 Functional Category enzyme ligaze Cell Location membrane associated intracellular OSTIGST 0ST 146301 Original Accession Mouse Nucleotide Accession NM 028817 Human Nucleotide Accession HM 004457 Summary The disrupted mouse gene is Acel3 acy LCoA synthetase long chain family member 3 ortholog of human ACSL3 Aliases include 2610510812Rik ACS3 PRO2194 FACL3 and lignoceroy CoA synthase Figure 18 Curated Gene Summary Information The Gene Summary section contains a curated description of each mutated gene in the database Most of the information is collected automatically then reviewed and curated independently by scientists This section contains information
9. 7 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation The CATScan page is rich in graphics and additionally provides the option to visualize a movie of the whole mouse CATScan Such movie can be saved on disk or be viewed using Windows Media Player plug in Results Micro CAT Data for F 98 ID Mouse Label Sex Genotype Mating 98 F 93 F hom 24643 Id represents the mouse number relative to this project Summary No abnormalities seen in gross skeleton thorax and abdomen Liver kidneys and spleen appear normal in size shape and position The rate of excretion of contrast media by the kidneys is within normal limits reflecting normal function Slicing Animation and 3D Projections Projection 3D body OONODOOO Slicing animation Launch Player Annotated Reference Movie Figure 15 CATScan Data Mouse Report An annotated mouse CATScan reference movie is also available Gallbladder YViONODOOO del Figure 16 Annotated Reference CATScan Movie f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Project Summary Page The Project Summary Page consists of six main sections Curated gene summary 2 Phenotypic Summary 3 Curated Mouse Gene Sequence Information 4 Curated Human Gene Sequence Information 5 Mutation Information 6 Vector Graphic From the Pr
10. Lexicon Phenotypic Analysis EEE __ x S i Li ae f Lexicon User Manual Basic Research Version May 2007 sai Lai A a LT t Se ae pp Saf pono I r i i mi cm cm lol s an cin m la Som fin ni ci Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Sofware Documentation Trademarks Lexicon Phenotypic Analysis Software and Database OmniBank Lexicon Pharmaceuticals Lexicon Pharmaceuticals are trademarks of Lexicon Pharmaceuticals Inc and may be registered in the United States or in other jurisdictions including internationally Other product names logos designs titles words or phrases mentioned within this publication may be trademarks servicemarks or tradenames of Lexicon Pharmaceuticals Incorporated Lexicon or other entities and may be registered in certain jurisdictions including internationally This document may contain links or references to third party web sites or publications that are not under the control of Lexicon Pharmaceuticals Inc and Lexicon is not responsible for the content of any such link or reference If you access a third party Web site mentioned in this guide then you do so at your own risk Lexicon provides these links only as a convenience and the inclusion of the link does not imply that Lexicon endorses or accepts any responsibility for the content on those third party sites Lexicon D
11. Software provides an easy way to compare data from different sets of animals based on their genotype The graphs are represented either as dot plots bar graphs or line graphs On each of the graphs throughout the software colors define the group of animals of a particular genotype for a particular mutation green for wild type animals blue for heterozygous animals and red for homozygous animals Note that the colors for the different genotype groups are the same in all graphs Below are a few examples of such graphs as they are displayed throughout the software Dot Plots BLOOD DATA BUN mg dL RSin 4 4 RSG 21 50 40 4 4 22 3047 204 HET BUN mg dL Figure 2 Dot Plot Graphs Standard Dew This is a typical dot plot graph showing data for a Blood Chemistry Test performed on 16 animals 4 wt in green 4 het in blue and 8 hom in red as shown in the line starting with RS n above the graph The RS line above the graph indicates either the mean or the median value for each group of animals In this example the mean values are 21 22 and 22 for wt het and hom distributions respectively The light green background indicates the Wild Type Range for all collected wild type range values By clicking on any graph throughout the software an extra window will open showing all available statistics that were computed for this particular Dataset Most of the standar
12. XP 32MB of RAM 64MB recommended a color monitor capable of 800x600 resolution 1024x768 recommended Microsoft Internet Explorer 6 0 or later version Firefox 1 0 6 or later version e For the Macintosh A Power Macintosh G3 or higher recommended running Mac OS X 10 3 or later 32MB or RAM 64MB recommended a color monitor capable of 800x600 resolution 1024x768 recommended Safari Browser Version 1 2 or later e Windows Media Player Plug in can be obtained from Microsofts web site Additional Required Software for Users No additional software is required for users to access the Lexicon Phenotypic Analysis Software other than a web browser as listed above Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved
13. an average value is computed and displayed on the graph for each distribution wt het and hom Again the green shaded background represents the ranges for all wild type values from previously collected Lexicon Phenotypic Analysis Projects for this Assay Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Lexicon Phenotypic Analysis Project Visualization Project Tree View The Project Tree View initially shows all available gene information for a selected Lexicon Phenotypic Analysis Project with a navigation tree to the left In this tree the first three links are the Gene Summary and the Genetics Report for this project The tree also lists all available Therapeutic Sections which include subfolders for all specific assays that were run for this project The figure below shows the Project Tree View interface Lexicon Lexicon Phenotypic Analysis LEXKO 011 B Project Summary E Genetics Gene Summary for LEXKO 011 Blood_Chemistry Functional Category enzyme ligase Cardiology Cell Location membrane associated intracellular Diagnostics OSTIGST OST 148301 Immunology Original Accession Metabolism Mouse Nucleotide Accession NM_028817 Neurology Human Nucleotide Accession NM 004457 Oncology Ophthalmology Pathology Expression E BEE TDR CESE Summary The disrupted mouse gene is Acsi3 acyl CoA synthetase long chain fami
14. ctor Graphic Vector Graphic VIC TR48 Total Size 5 174 nucleotides Non Cutters Apal XhoI Xmnl Unique sites Kpnl 2671 HindIII 847 Sacl 2677 vac 178 Boll 754 Sacl 1692 sacl 3595 Kpn 4695 Kpnl 111 Sacli2712 Bents60l 32014762 LTE sa NEO pA S8V40tpA PGE BTESD LTE The vector graphic displays detail regarding the retroviral vector used to generate a knockout mouse via retroviral insertion mutagenesis This section is not displayed for knockout lines generated by homologous recombination f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Genetics Analysis Report The Genetics Analysis Report summarizes the genotype distributions and statistics for wild type heterozygous and homozygous animals in each Lexicon Phenotypic Analysis Project This information is used to infer embryonic lethality or reduced viability f Lexicon LEXKO 011 D Project Summary Genetics Blood Chemistry Cardiology Diagnostics 1 Immunology i Metabolism 1 Neurology Oncology l Ophthalmology Pathology Expression Lexicon Phenotypic Analysis Genotyping Strategy Genetics Information Targeted or gene trap mutations are generated in strain 129SvEv _derivedembryonic stem ES cells The chimeric mice are bred to CS7BL 6J albino mice to generate F1 heterozygous animals These progeny are intercrossed togenerate F2 wild typ
15. d Statistical methods have been used and are made available to the user for each genotype group BLOOD DATA BUN mg dL Overall WwT i mean amp standard deviation te Sx 0 i i ct tean dn de St Mi O O O CE OO SO HET Js Eroso rezza possi a a fr Fosso zvoc foo Jaan Jr fo HOM J 21000 frets cozze sec foo fr eee reo foo for fea f Lexicon Figure 3 Available Statistical Indicators for all Graphs Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Also note that throughout the software there are Lexicon Phenotypic Analysis Software Documentation blue bars at the top and bottom of each graph BLOOD DATA ALK_PHOS U L These bars represent the usual values for a typical assay Such ranges are used to ensure consistency in the drawing of graphs from project to project for the same assay In some cases where outliers are present outside of this range test failure extreme values etc the graph will display the outlier s and scale the graph accordingly In effect the blue bars will now be displayed in the middle of the figure to indicate which value s seems to be outside the normal range Figure 4 illustrates an example of such an occurrence Bar Graphs URINAY SIS Nitrite Lul T HET HOM f E di WT HET HOM Figure 5 Bar Graphs Lexicon RSin 4 4 RSG 124 HET HM ALK PHOS U L Figure 4 Scaling of graphs
16. e heterozygous and homozygous mutant progeny On rare occasions for example when very few F1 mice are obtained from the chimera F1 heterozygous mice are crossed to 129SvEv C57 hybrid mice to yield additional heterozygous animals for the intercross to generate theF2 mice Level phenotypic analysis is performed on mice from this generation wt het hom Total Observed 9 40 14 63 Expected 15 75 31 5 15 75 63 Chi Sq 3 09 Significance 0 21331188 hom n 0 23 Avg Litter Size 9 Copyright 2007 by Lexicon Pharmaceuticals Incorporated Lexicon Phenotypic Analysis software version 07 05 00 00 Figure 22 Genetics Analysis Report A link to a PDF file detailing the Genotyping Strategy is also available The figure below shows an example of the contents of such a file LEXKO 011 Genotyping Strategies Reaction Components Vol full Temp i e 1 0 eee gt rc 40 Goto5 HI ge otal reaction volume Recommended Wt Oligos LEXKO 011 5 wt and LEXKO 01 1 3 464 bp LEXKO 011 5 wi Mutant PCR We Sample Genotype 2 3 4 5 MK o es L hm waster _ no amp 2 Agarose NEB 100 bp ladder Figure 23 Genotyping Strategy Available Information if Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Phenotypic Assays amp Data As described previously 10 Therapeutics Sections are used t
17. ent has occurred These types of events are engineered to completely ablate the function of the gene and result in a null mutation Targeting vectors usually contain a reporter gene typically LacZ which may be used to determine the temporal and spatial expression patterns of the gene targeted Gene Trapping Gene Trapping provides a high throughput approach for producing insertional mutations in genes When gene trapping is performed in mouse embryonic stem cells these cells can subsequently be used to produce mutant mice Gene Trapping vectors are delivered to embryonic stem cells by infection with an engineered retrovirus Upon entry into the cell the i Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation vector randomly inserts into the genomic DNA inserting into and mutating genes The vectors are designed so that insertion events occurring in transcriptionally active areas result in the expression of a selectable marker gene thereby enriching for recovery of insertion events within genes Subsequent selection allows the growth of stem cell colonies from clones containing a single gene trap insertion event The gene trap event not only mutates the gene but it also marks the gene by virtue of insertion of unique genetic elements making it possible to identify the sequence of the mutated gene using a variety of PCR based techniques Conditional Mutati
18. et O 057 O5654 34 75 25 09 2547 22 2 18 09 0 065 0 097 70000 M 11 4 0 0499 0 4961 26 33 11 92 22 7 21 66 15 02 0 0595 007625000 M 1 25 0 0561 0 635 31 93 24 05 25 62 26 59 157 C 0752 009250000 M 1 24 0 0543 0 5676 20 01 16 56 22 06 24 63 16 79 0 0636 009075000 M 1 40 O 06 O 6617 36 53 24 65 26 06 24 65 18 07 0 0695 009445000 DEXA Analysis Female Whole Body Vertebrae Femurs Mouse Label GeNOtype BMD Bac x rem PMC vemp BMD BMD gicm 9 TIM FAT u mgicm igicm g em mgg F 127 rt 0 052 0 461 20 7 15 99 76 91 29 45 17 1 0 0615 007505000 F 97 wt 004r 0 507 21 59 15 32 17 70 20 52 15 1 0 067 007205000 F 118 het 0 0506 0 5021 23 76 19 09 18 53 27 09 16 15 O 0712 007395000 F 125 het O 0537 05405 23 63 17 24 19 02 20 42 16 93 O 0714 007520000 F 117 0 0512 0 5238 21 65 15 4 17 62 294 16 01 0076 007505000 F 126 00525 0 5119 20 79 14 15 17 33 29 54 16 5 0 0655 007430000 F 89 0 0501 0 4954 24 53 20 0 18 93 26 17 15 92 0 07 0 07445000 F 95 0 0521 0 5078 23 75 23 41 17 60 26 72 16 69 OOF O 0FS50000 Figure 12 Another Example of Calculated Data Results f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation DEXA Analysis data Mouse Label Genotype Age days Site BMD BMC Length Weight FAT TIM F 116 het 106 T Animal Total 0 0508 0 5021 9 5 25 393 19 69 25 76 F 119 het 106 R FL 075 0 0294 9 5 25 30 24 52 DE F 119 het 106 R FR 0 0722 0 0292 dI 25
19. he phenotypic information and validated data associated with large scale analysis of knockout mice Using a convenient web interface users can assess and search a wide range of phenotypic information for each gene knockout project including raw data calculated graphs with annotated summaries and statistical analysis for each of the major therapeutic areas also referred to as Therapeutic Sections in this document This document provides users with a means of becoming familiar with this basic research version of the Lexicon Phenotypic Analysis Software and learning how to use all of its features in order to assess and retrieve phenotypic information and data from gene knockout projects Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Terminology used in this manual Several defined terms are used throughout the Lexicon Phenotypic Analysis Software and deserve particular attention to understand how they are referenced in this document and the associated software Lexicon Phenotypic Analysis Project Throughout the Lexicon Phenotypic Analysis Software each genetic mutation for every target gene is presented as a single Project and there are many ways of accessing and retrieving data for the mouse knockout projects that may be of greatest interest to research scientists The same gene can be mutated using several different strategies Gene
20. ic graphs or data transformations are often necessary in order to interpret the raw data and the software is pre configured to perform these operations natively For example Open Field Tests may initially generate trace information over a fixed period of time and the software automatically transforms such data into different outputs such as Total Distance or Center vs Total Distance Ratio Assay Values In most cases a single Assay can produce multiple pieces of information or datapoints in one experiment For example a typical Hematology Test will produce as many as 20 different Assay Values such as Red Blood Cell Count White Blood Cell Count Glucose Level etc The software automatically graphs such values independenily yet retains the original raw data information structure Such Assay Values can also be independently analyzed based on specific built in criteria For instance gender may be an important variable in certain experiments hence the software will provide combined data graphs as well as separate graphs from male and female data Datasets The notion of Datasets is one of the most important and powerful features of the Lexicon Phenotypic Analysis Software Datasets are generated from different scientific or experimental protocols and they are used to keep such data in different bins for proper interpretation If two different scientific protocols were used for the same assay combining the results could impact their scie
21. isclaimer LEXICON PHARMACEUTICALS INC MAKES NO WARRANTIES EITHER EXPRESS OR IMPLIED REGARDING THE ENCLOSED COMPUTER SOFTWARE PACKAGE ITS MERCHANTABILITY OR ITS FITNESS FOR ANY PARTICULAR PURPOSE THE EXCLUSION OF IMPLIED WARRANTIES IS NOT PERMITTED BY SOME STATES THE ABOVE EXCLUSION MAY NOT APPLY TO YOU THIS WARRANTY PROVIDES YOU WITH SPECIFIC LEGAL RIGHTS THERE MAY BE OTHER RIGHTS THAT YOU MAY HAVE WHICH VARY FROM STATE TO STATE Copyright 2007 Lexicon Pharmaceuticals Inc All rights reserved This manual may not be copied photocopied reproduced translated or converted to any electronic or machine readable form in whole or in part without the prior written approval of Lexicon Pharmaceuticals Inc Lexicon Pharmaceuticals Incorporated 8800 Technology Forest Place The Woodlands TX 77381 1160 USA http www lexpharma com Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Introduction These web pages have been generated by Lexicon Phenotypic Analysis Software and Database an online analytical and decision support system that catalogs the in vivo characterization of all genes that are knocked out using either gene targeting or gene trapping technologies This software is a module in a much broader package that includes the Lexicon Mouse Production Software the Lexicon Phenotypic Data Collector Software and the Lexicon Phenotypic Data
22. listed Assays in this section is named Blood Chemistry and contains a specific Dataset named Results that provides a Data Page for this set of data The next picture describes a typical Project Tree View page if the user clicks on the Results Dataset link for the LEXKO 011 Lexicon Phenotypic Analysis Project f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation if Lexicon Lexicon Phenotypic Analysis LEXKO 011 B Project Summary Blood Chemistry for Project LEXKO 011 z Genetics Test Description Lexicon Genetics uses the COBAS Integra 400 Blood Chemistry mfr Roche in its clinical settings for running blood chemistry E Blood Chemistry tests on mice Results HgbA1C Insulin Oral_Glucose Tolerance Urinalysis Cardiology Diagnostics Immunology Metabolism Level Neurology Oncology ALK PHOS ALKaline PHOSphatase ALT ALanine AminoTransferase m ALB2 Albumin BILT Bilirubin Total Pathology _ 6 Expression CHOL Total CHOLesterol PHOS PHOSphorous TRIG TRIGlyceride CREAT CREATinine BUN Blood Urea Nitrogen CALCIUM CALCIUM GLUCOSE GLUCOSE UA2 Uric Acid SODIUM SODIUM POTASSIUM CHLORIDE CHLORIDE Ae A E ALK_PHOS Female Only id File Based Data Ph 150 125 Standard Data File Based Data ame Standard Data 75 Figure 8 Project Tree View Navigation Access
23. ly member 3 ortholog of human ACSL3 Aliases include 2610510B12Rik ACS3 PRO2194 FACL3 and lignoceroy CoA synthase Phenotypic Summary LEXKO 011 The homozygous mutant mice exhibited increased peripheral blood percentages of CD4 cells and decreased B cells when compared with their wild type littermates and the historical means Decreased IgG1 response to ovalbumin was noted in mice In addition the male mutants exhibited signs of growth retardation including decreased mean body weight and length total tissue mass and lean body mass E RT PCR analysis revealed that the transcript was absent in the homozygous mutant mice Mouse Gene Sequence Information Nucleotide NM 028817 ACCESSION NM_028817 NID gi 75992919 ref NM_028817 2 Mus musculus acyl CoA Reference synthetase long chain family member 3 Acsl3 transcript variant 1 mRNA Protein Reference Entrez Gene 74205 Human Gene Sequence Information 2 Nucleotide NM 004457 ACCESSION NM_004457 NID na Homo sapiens Homo sapiens fatty acid Coenzyme A Reference ligase long chain 3 FACL3 mRNA hs_refseq Q9CcZW4 Q9CZW4 Q9CZW4 2610510B12RIK PROTEIN File Based Data Protein Standard Data Reference E File Based Data Entrez Gene 2181 Standard Data Mutation Information Figure 7 Project Tree View Page 095573 LCFC_HUMAN 095573 LONG CHAIN FATTY ACID COA LIGASE 3 EC By clicking on the Blood Chemistry Therapeutic Section one of the
24. ntific interpretation For example if some mice are subjected to a high fat diet before a Blood Chemistry test is performed the results should not be combined with results from mice subjected to a normal chow diet In this particular case two datasets are created one for the High Fat Diet Protocol and one for the Chow Diet Protocol Different Datasets are used when experimental conditions change for a particular assay over time changing environmental conditions such as water equipment etc In such cases and since data variations may prevent different projects from being compared against one another it is often beneficial to create a new dataset that will be used to store the newly created data Different Datasets are also used when animals are subjected to or challenged with specific drugs or agents prior to being analyzed through the available phenotypic Assays Throughout these pages a single Dataset is being used and is named Results Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Wild Type Historical Data Wild Type Ranges An additional reason Datasets are created for each protocol is that the software can produce Wild Type Historical data or ranges by combining wild type Assay Values for many projects studied under the same experimental protocol This allows computational analysis using large wild type control populations fo
25. o classify all the Assays available for a Lexicon Phenotypic Analysis Project i e mutated gene Blood Chemistry Therapeutic Section Data may be available for the following Assays in this section Blood Chemistry graphs data HGBA1C graphs data Insulin Assay graphs data Glucose Tolerance graphs data Oral Glucose Tolerance graphs data Urinalysis graphs data Cardiology Therapeutic Section Data may be available for the following Assays in this section e Blood Pressure graphs data e Heart Rate graphs data Diagnostics Therapeutic Section Data may be available for the following Assays in this section e Fertility data e Length graphs data e Weight graphs data Expression Therapeutic Section Data may be available for the following Assays in this section Contig Map images QC Expression images QC Images images sequences WT Panel images Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Immunology Therapeutic Section Data may be available for the following Assays in this section Acute Phase Response graphs data FACS Analysis graphs data Ovalbumin Challenge graphs data Peritoneal Inflammation Assay data Serum Immunoglobulin Assay graphs data sICAM 1 Assay graphs data Metabolism Therapeutic Section Data may be available for the following Assays in this section
26. oject View Page Tree clicking on the Gene Summary link will display a page as shown in the figure below if Lexicon LEXKO 011 D Project Summary Genetics El Blood Chemistry E Cardiology Diagnostics Immunology Metabolism Neurology Oncology Ophthalmology H Pathology E Expression File Based Data E Standard Data File Based Data Standard Data if Lexicon Lexicon Phenotypic Analysis Gene Summary for LEXKO 011 Functional Category enzyme ligase Cell Location membrane associated intracellular OST GST OST 148301 Original Accession Mouse Nucleotide Accession NM 028817 Human Nucleotide Accession NM_004457 Summary The disrupted mouse gene is Acsl3 acyl CoA synthetase long chain family member 3 ortholog of human ACSL3 Aliases include 2610510B12Rik ACS3 PRO2194 FACL3 and lignoceroy CoA synthase Phenotypic Summary LEXKO 011 The homozygous mutant mice exhibited increased peripheral blood percentages of CD4 cells and decreased B cells when compared with their wild type littermates and the historical means Decreased IgG1 response to ovalbumin was noted in mice In addition the male mutants exhibited signs of growth retardation including decreased mean body weight and length total tissue mass and lean body mass RT PCR analysis revealed that the transcript was absent in the homozygous mutant mice Mouse Gene Sequence Information 2 Nucleotide NM 028317 ACCESSION NM_
27. on Conditional Mutations make use of site specific recombinases e g Cre that delete genomic sequence between their recognition sites e g LoxP To produce a conditional mutation a region of a gene is flanked by LoxP sites using homologous recombination in ES cells The LoxP sites along with a selectable marker are positioned in non coding regions of the gene and by design should not themselves affect expression of the gene By crossing these mice with a line of mice that expresses Cre recombinase in a specific tissue and or at a specific time one can generate temporal or tissue specific mutations as the gene will only be deleted where and when the recombinase is expressed Mouse Label A Mouse Label is a unique identifier for an animal that is constructed from a single capital letter indicating the mouse gender and a unique serial number for this mouse within this project For example a Mouse Label noted M 100 within a Lexicon Phenotypic Analysis Project refers to the 100th mouse a male M bred within this project Mouse Genotype Each animal carries a mandatory Mouse Genotype designation as hom homozygous het heterozygous or wt wild type for each mutation Phenotypic analysis is generally performed by comparing assay values in a Lexicon Phenotypic Analysis Project between genetically matched cohorts of mice The Lexicon Phenotypic Analysis Software allows for determining a gene specific knockout phenotype by comparing the data
28. on provides a link to public sequence records for the mutated mouse gene along with the gene s description Also included is a link to NCBI or the SwissProt database for the mouse protein sequence with its definition and finally a link to the Entrez Gene record is displayed Human Gene Sequence Information Human Gene Sequence Information Nucleotide NM 004457 ACCESSION Nh 004457 NID na Homo sapiens Homo sapiens fatty aci Coenzyme A Reference ligase long chain 3 FACL3 mRNA hs_refseg Protein Reference Entrez Gene 2181 095573 LCFC_HUMNAN 095573 LONG CHAIN FATTY 4 CID COA LIGASE 3 EC Figure 20 Human Gene Sequence Information This section provides a link to public sequence records for the human gene ortholog to the mutated mouse gene along with the human gene s description Also included is a link to NCBI or the SwissProt database for the human protein sequence with its definition and finally a link to the Entrez Gene record is displayed Gene Mutation Information Mutation Information Mutation Type Retroviral Insertion OST Description Retroviral insertion disrupted the gene upstream of coding exon 1 AK012068 Figure 21 Mutation Information The Mutation Type and a short description of the mutation strategy are described in this section of the Gene Summary Page f Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Ve
29. otal Tissue Mass g BMC LBM BMC LBM mg g vBMD Volumetric BMD mg cm MALE DATA DESA M Total Body TTM DEXA M LEM DEXA M Total Body FAT oats 29 9 aida abita W 2885 igo 1746 2983 WB 15 87 18 6 40 00 sona gl Lvi w o wW wm sa Sio so SEO 0 dse daa dab CBS Wo 2454 1883 odi 2317 is aaa 35 00 12 00 0 70 an nn Figure 10 DEXA Assay Graphs Lexicon Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation DEXA Combined Gender Score Parameter Score Body BID 107 2 Body vBMD 105 4 Body BMC LAr 104 1 Femur BMD 106 4 Vertebral BMD 105 4 Total Tissue Mass 109 2 Body LBM 106 0 Body Fat 111 4 Body Fat Content 124 4 The Combined Gender Score provides the average ration of Horm to WNT values combining males and females with a correction factor for the number of mice in each group No score is generated if there are fevver than 4 HOM mice or fever than 3 WNT mice Figure 11 An Example of Calculated Data Results DEXA Analysis Male Whole Body Vertebrae Femurs dia Genotype BMD BMC cw i LBM BMC BMD BMD BMD gemf 9 FAT g mali mgicm gemf giem bl 113 wt 0 0499 0 5251 27 96 14 68 23 33 22 51 14 58 0 0593 D 08090000 M 119 wt O 0503 04771 27 34 21 96 20 86 22 07 16 34 O lt 0576 007835000 bl 131 het 0 0504 0 5334 20 59 14 96 24 02 22 21 15 49 0 09 0 0641 5000 M 139 h
30. r statistical analysis The version of the Lexicon Phenotypic Analysis Software used to generate these pages was configured to only provide overall historical wild type ranges for each assay dataset graph combination These ranges have been generated from thousands of wild type datapoints from projects having already been fully phenotyped by Lexicon Pharmaceuticals On each graph the data for the different genotype distributions wt het and hom will be displayed along with a representative set of historical wild type values from other projects whose data was previously collected under the same experimental conditions i e Dataset The overall historical range of values for all previously collected data points from wild type animals is displayed as a green shaded background with two tones of green representing the range for one standard deviation around the mean in light green color 1 and two standard deviations in darker green color 2 Additional information provided for all available graph types is available in more detail later in this document i Lexicon BLOOD DATA CHOL mg dL RS n 4 4 4 4 6 6 RSI 102 120 129 HET CHOL mg dL Figure 1 Wild type Historical Ranges Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation Graphs amp Calculated Data Available Graphs in Lexicon Phenotypic Analysis Software The Lexicon Phenotypic Analysis
31. to all available data for an Assay All data available for an Assay will be displayed on the data page along with e adescription of the Assay and protocol used when relevant e all graphs in different conditions when necessary e alink to a csv file with all raw data for animals analyzed through this assay In some cases additional calculated data tables are available providing the scientist with additional data analyses used to assess a phenotype more efficiently or practically than when using the raw data The figures below show the contents of a data page for the DEXA Assay Copyright 2007 Lexicon Pharmaceuticals Incorporated All Rights Reserved Lexicon Phenotypic Analysis Software Documentation DEXA for Project LEXKO 011 Test Description Mice were anesthetized by intraperitoneal Injection of Avertin 1 25 2 2 2 tribromoethanol 20 ml kg body weight measured body length and weight and then placed in a prone position on the platform of the PIXimus Densitometer Lunar Inc for a DEXA scan Using a Lunar PIXImus software the Bone mineral density BMD and fat composition fat and total tissue mass TTM were determined in the regions of interest ROI i e whole body vertebrae and both femurs Figure 9 Description of the DEXA Assay Level BMD Bone Mineral Density q cm FAT of Body FAT BMC Bone Mineral Content g FAT g TIM X FAT 100 LBM Lean Body Mass g TTM T
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