WormBase Model:Construct
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Contents
Proposed model changes
Purpose: the technology of engineering mutations and gene replacement has been developed in C. elegans. With these new research tools, capture and display of the molecular information of these alleles needs to be updated. We propose a new class, Construct, to capture the specifics of the DNA tool used to perform the replacement or engineering, while the Variation model gets updated to record the engineering event itself and its impact on the genome. As a side benefit to the creation of the Construct model, we can use this new class to also capture the genomic arrays used create transgenes.
Variation
Proposed additions
?Variation
Variation_type
Engineered_allele
Variation_summary ?Text//to house final engineered construct
Derived_from ?Construct XREF Engineered_variation
Corresponing_transgene Unique ?Transgene XREF Corresponding_variation
Method
Homologous_recombination //Homologous_recombination
NHEJ //Non-homologous DNA end-joining, imprecise DNA repair
MosSci
TALENs
CRISPR_Cas9
ZFN-NHEJ repair //Zinc-finger nuclease
ZFN-HR repair
Expr_pattern ?Expr_pattern XREF Variation #Evidence
notes on variation model changes
see discussion tab
WBPaper00045772 Paix et al., 2014
Discusses Homology directed repair (HDR) vs Nonhomologous end-joining (NHEJ). It would seem that distinguishing the method used to create engineered alleles is important. However, the tags should perhaps be modified to distinguish double stranded break method and repair method.
break_made_by Mos_excision CRISPR/Cas9 TALENs Zinc_finger_endonuclease OTHER repaired_by nonhomologous_end_joining_NHEJ homologous_direct_repair_HDR
--Kyook (talk) 21:17, 16 October 2014 (UTC)
Construct (new)
Class itself is new
?Construct Evidence #Evidence Curator_confirmed ?Person Public_name ?Text Other_name ?Text Summary ?Text //genotype like [Pmyo-2::GFP] Sequence_feature ?Feature XREF Associated_with_construct Driven_by_gene ?Gene XREF Drives_construct #Evidence Gene ?Gene XREF Construct_product #Evidence 3_UTR ?Gene #Evidence Fusion_reporter ?Text //fluorescent proteins GFP, RFP, mCherry, etc. Other_reporter ?Text //to add reporters, tags that aren�-F¢t included in model�-A Purification_tag ?Text //FLAG, HA, Myc, TAP, etc. Recombination_site ?Text //LoxP, FRT Type_of_construct Chimera Domain_swap Engineered_mutation Fusion Complex // complex changes Transcriptional_fusion Translational_fusion Nterminal_translational_fusion Cterminal_translational_fusion Internal_coding_fusion Selection_marker ?Text Construction_summary ?Text // Backbone vector, mol bio DNA_text Text // for mapping, can include entire construct sequence Clone ?Clone XREF Construct Used_for Transgene_construct ?Transgene XREF Construct Transgene_coinjection ?Transgene XREF Coinjection Engineered_variation ?Variation XREF Derived_from_construct Topic_output_indicator ?WBProcess XREF Marker_construct Expression_pattern ?Expr_pattern XREF Construct Interactor ?Interaction Reference ?Paper Person ?Person Laboratory ?Laboratory #Lab_Location Historical_gene ?Gene #Evidence Remark ?Text #Evidence
notes on construct model
see discussion tab
#Interactor_info
Construct ?Construct XREF Interactor
Interaction
Unaffiliated_construct ?Construct Detection_method Construct
Expression_pattern
proposed addition
Variation ?Variation XREF Expression_pattern Construct ?Construct XREF Expression_pattern
Gene
proposed change
Drives_Transgene ?Transgene XREF Driven_by_gene change to Drives_construct ?Construct XREF Driven_by_gene
Transgene
Proposed changes: Many of the transgene tags have been moved to the proposed ?Construct model, the remaining tags as well as some additions are shown below
?Transgene Evidence #Evidence
Public_name UNIQUE ?Text
Summary UNIQUE ?Text
Synonym ?Text
Corresponding_variation UNIQUE ?Variation XREF Corresponding_transgene //put in to unambiguously associate the allele/transgene
Construction Construct ?Construct XREF Transgene_construct
Coinjection ?Construct XREF Transgene_coinjection
Coinjection_other ?Text //for coinjection markers that are not specified as a construct
Integration_method UNIQUE ?Text
Integrated_from ?Transgene XREF Transgene_derivation
Laboratory ?Laboratory #Lab_Location
Author ?Author
Construction_summary ?Text
Genetic_information Extrachromosomal
Integrated
Map ?Map #Map_position //needed for transgenes with no granular mapping, e.g., just mapped to a LG
Mapping_data 2_point ?2_point_data //deleted for WS245, rolled back for WS246
Map_evidence #Evidence
Phenotype ?Phenotype XREF Transgene #Phenotype_info
Phenotype_not_observed ?Phenotype XREF Not_in_Transgene #Phenotype_info
Used_for Transgene_derivation ?Transgene XREF Integrated_from
Expr_pattern ?Expr_pattern XREF Transgene
Marker_for ?Text #Evidence
Interactor ?Interaction
Associated_with Marked_rearrangement ?Rearrangement XREF By_transgene
Strain ?Strain XREF Transgene
Reference ?Paper XREF Transgene
Species UNIQUE ?Species
Remark ?Text #Evidence
Historical_gene ?Gene #Evidence
WBProcess
Marker_construct ?Construct XREF Topic_output_indicator
Test data
Variation objects with constructs
LP132 nmy-2(cp7[nmy-2::gfp + LoxP unc-119(+) LoxP]) I; unc-119(ed3) I
Variation : "WBVar020000000" Public_name "cp7" Engineered_allele Variation_summary "[nmy-2::gfp + LoxP unc-119(+) LoxP]" Derived_from "WBConstruct00000010" Derived_from "WBConstruct00000011" Homologous_recombination Construct : "WBConstruct00000010" Summary "[nmy-2::gfp] Driven_by_gene "WBGene00003777" Fusion_reporter "GFP" N-terminal_translational_fusion Construct : "WBConstruct00000011" Summary "[LoxP unc-119(+) LoxP] Recombination_site "LoxP" Gene "WBGene00003777"
bus-50(e5000[T110E]) = An engineered missense mutation
bus-50(e5001[bus-50::gfp]) aka bus-50::gfp = An engineered fusion of GFP to the C-terminus of BUS-50
Variation : "WBVar0200000001" Public_name "e5001" Engineered_allele Variation_summary "[bus-50::gfp]" Derived_from "WBConstruct00000012" CRISPR-Cas9 Construct : "WBConstruct00000012" Summary "[bus-50::gfp]" Gene "WBGene0020000001" Fusion_reporter "GFP" C-terminal_translational_fusion
bus-50(e5002[bus-50::gfp + loxP unc-119(+) loxP]) An engineered insertion of GFP plus the unc-119(+) selectable marker, flanked by loxP sites.
Variation : "WBVar020000003" Public_name "e5002" Engineered_allele Variation_summary "[bus-50::gfp + loxP unc-119(+) loxP]" Derived_from "WBConstruct00000012" Derived_from "WBConstruct00000011" HR Construct : "WBConstruct00000012" Summary "[bus-50::gfp]" Gene "WBGene0020000001" Fusion_reporter "GFP" C-terminal_translational_fusion Construct : "WBConstruct00000011" Summary "[LoxP unc-119(+) LoxP] Recombination_site "LoxP" Gene "WBGene00003777"
bus-50(e5003[bus-50::gfp +loxP]) aka bus-50(e5003) = bus-50(e5002) following Cre-mediated recombinase removal of unc-119(+) leaving a single loxP site
Variation : "WBVar020000004" Public_name "e5003" Engineered_allele Variation_summary "[bus-50::gfp + loxP]" Derived_from "WBConstruct00000012" Derived_from "WBConstruct00000011" HR Construct : "WBConstruct00000012" Summary "[bus-50::gfp]" Gene "WBGene0020000001" Fusion_reporter "GFP" C-terminal_translational_fusion Construct : "WBConstruct00000011" Summary "[LoxP unc-119(+) LoxP] Recombination_site "LoxP" Gene "WBGene00003777"
Variation object with transgene
eIs2002 = eIs2002[unc-119::gfp] = eIs2002[unc-119::gfp, III:2992500] Engineered insertions in apparent intergenic region with optional descriptors (nature of the insertion or position in the genome)
Variation : "WBVar020000005" Public_name "eIs2002" Engineered_allele Variation_summary "[unc-119::gfp]" Derived_from "WBConstruct00000013" Identical_transgene "WBTransgene00024514" MosSci Construct : "WBConstruct00000013" Summary "[unc-119::gfp]" Gene "WBGene0020000001" Fusion_reporter "GFP" Translational_fusion Transgene : "WBTransgene00024514" Public_name "eIs2002" Summary "[unc-119::GFP]" Construct "WBConstruct00000013"
ozIs909, or ozIs909[unc-119::mCherry *eIs2002] = Engineered changes to existing Is (or Si) insertions, which should receive new Is numbers using originating lab’s prefix. The original Is insertion is indicated in brackets with a preceding asterisk (*), in order to allow searches for all derivatives from a given insertion. In this case, an engineered change from GFP to mCherry in eIs2002
Variation : "WBVar020000006" Public_name "ozIs909" Engineered_allele Variation_summary "[unc-119::mCherry *eIs2002]" Derived_from "WBConstruct00000015" Identical_transgene "WBTransgene00024515" MosSci Construct : "WBConstruct00000015" Summary "[unc-119::mCherry]" Gene "WBGene0020000001" Fusion_reporter "mCherry" Translational_fusion Transgene : "WBTransgene00024515" Public_name "ozIs909" Summary "[unc-119::mCherry] Construct "WBConstruct00000015"
Transgene with construct
The following depicts how current transgenes would be redistributed into the proposed Construct and Transgene models
(Original)Transgene : "WBTransgene00000001" Public_name "adEx1256" Summary "[egl-19::sGFP-NLS + lin-15(+)]" Reporter_product "GFP" Driven_by_gene "WBGene00001187" Strain "DA1256" Reference "WBPaper00029359" Reporter_type "Transcriptional fusion" Synonym "[C48A7.1::gfp]" (New)Transgene : "WBTransgene00000001" Public_name "adEx1256" Summary "[egl-19::sGFP-NLS + lin-15(+)]" Construct "WBConstruct00000001" Coinjection_other "lin-15(+)" Extrachromosomal Strain "DA1256" Reference "WBPaper00029359" (New)Construct : WBConstruct00000001 Public_name "adEx1256" Other_name "[C48A7.1::gfp]" Summary "[egl-19::sGFP-NLS]" Fusion_reporter "GFP" Driven_by_gene "WBGene00001187" Reference "WBPaper00029359" Transcriptional_fusion
(Original)Transgene : "WBTransgene00000011" Public_name "adIs1240" Summary "[lin-15(+) eat-4::sGFP]" Reporter_product "GFP" Driven_by_gene "WBGene00001135" Strain "DA1240" Strain "DA1243" Map "X" Map_evidence Paper_evidence "WBPaper00038205" Reference "WBPaper00030960" Reference "WBPaper00032252" Reference "WBPaper00035265" Reference "WBPaper00036277" Reference "WBPaper00036704" Reference "WBPaper00037626" Reference "WBPaper00038205" Reference "WBPaper00044482" Synonym "[eat-4::gfp]" (New)Transgene : "WBTransgene00000011" Public_name "adIs1240" Summary "[lin-15(+) eat-4::sGFP]" Strain "DA1240" Strain "DA1243" Map "X" Map_evidence Paper_evidence "WBPaper00038205" Coninjection_other "lin-15(+)" Reference "WBPaper00030960" Reference "WBPaper00032252" Reference "WBPaper00035265" Reference "WBPaper00036277" Reference "WBPaper00036704" Reference "WBPaper00037626" Reference "WBPaper00038205" Reference "WBPaper00044482" (New)Construct : "WBConstruct00000011" Public_name "adIs1240" Other_name "[eat-4::gfp]" Summary "[eat-4::sGFP]" Fusion_reporter "GFP" Driven_by_gene "WBGene00001135" Reference "WBPaper00030960"
(Original) Transgene : "WBTransgene00000017" Public_name "ajIs1" Summary "[pgp-5::gfp]" Coinjection_marker "pRF4[rol-6(su1006)]" Construction_summary "Integrated from BC10030 sEx864." Reporter_product "GFP" Driven_by_gene "WBGene00003999" Driven_by_gene "WBGene00006767" Integration_method "Gamma_ray" Integrated Reference "WBPaper00002968" Reference "WBPaper00031023" (New) Transgene : "WBTransgene00000017" Public_name "ajIs1" Integration_method "Gamma_ray" Integrated Reference "WBPaper00002968" Reference "WBPaper00031023" Integrated_from "WBTransgene00002030" Construction_summary "Integrated from BC10030 sEx864." (New) Transgene : "WBTransgene00002030" Public_name "sEx864" Synonym "[pgp-5::gfp]" Synonym "[C05A9.1::gfp]" Summary "[rCesC05A9.1::GFP + pCeh361]" Extrachromosomal Construct "WBConstruct00000017" Construct "WBConstruct00000018" Construct "WBConstruct00000002" (New) Construct : "WBConstruct00000018" Public_name "pRF4" Summary "[rol-6(su1006)]" Gene "WBGene00004397" (New) Construct : "WBConstruct00000017" Public_name "[pgp-5::gfp]" Summary "[rCesC05A9.1::GFP]" Reporter_product "GFP" Transcriptional_fusion Driven_by_gene "WBGene00003999" (New) Construct : "WBConstruct00000002" Public_name "pCeh361" Summary "[pCeh::DPY-5]" Clone "pGEM-5 Construction_summary "A 3.3-kb Nco I fragment containing a predicted cuticle collagen gene was isolated from the cosmid F27C1, and cloned into the Nco I site of pGEM-5 to generate the clone pCeh361" Reference "WBPaper00027361"
(New) Transgene : "WBTransgene00000600" Public_name "hIs2" Summary "[DPY-5::GFP + rol-6(su1006) + pBluescript] Construct "WBConstruct00000003"//pCeh358 Coinjection "WBConstruct00000004"//PCes1943 Coinjection "WBConstruct00000005"//pBluescript KS Construction_summary "Transgenic animals were generated by microinjection of pCeh358 (5 ng/ul) and pBluescript KS (100 ng/ul), or in combination with 50 ng/ul pCes1943, which carries a dominant rol-6 mutation [rol-6(su1006)] used as a morphological marker for successful transformation" (New) Construct : "WBConstruct00000003" Public_name "pCeh358" Summary "[dpy-5::gfp]" Driven_by_gene "WBGene00001067" Gene "WBGene00001067" Clone "pPD95.69" Translational_fusion Construction_summary "The dpy-5::gfp reporter construct pCeh358 was generated by insertion of a 750 bp Sph I fragment from pCeh361 into the Sph I site of the gfp expression vector pPD95.69 (kindly provided by A. Fire). This fragment contains 5' sequences from an Sph I site in the polylinker of pCeh361 to a site 30 bp downstream from the predicted DPY-5 initiator methionine, resulting in an in-frame fusion of the first 12 codons of dpy-5 with gfp." Reference "WBPaper00027361" (New) Construct : "WBConstruct00000004" Public_name "pCes1943" Summary "[rol-6(su1006)] (New) Construct : "WBConstruct00000005" Public_name "pBluescript KS"
Expression objects with construct
Examples of two expression objects pertaining to sequence feature
Example 1: Expr_pattern : "Expr11377" Anatomy_term "WBbt:0005813" Certain Anatomy_term "WBbt:0008588" Certain Anatomy_term "WBbt:0008589" Certain Gene "WBGene00001948" Life_stage "WBls:0000003" Life_stage "WBls:0000041" Pattern "enh-1 was preferentially active in the posterior C and D lineages in the embryo and in body wall musculature in the adult." Reference "WBPaper00032967" Reporter_gene Construct "WBConstruct00000020" Associated_feature "WBsf047531" Construct : "WBConstruct00000020" Public_name "enh-1 reporter" Summary "[hlh-1.enh-1::pPD107.94]" Clone "pPD107.94" //added clone back to the construct model Sequence_feature "WBsf047531" Gene "WBGene00001948" Fusion_reporter "GFP" Construction_summary "Enhancer region enh-1 for hlh-1 cloned into the (del)Pes-10 basal promoter vector pPD107.94" Reference "WBPaper00032967" Example 2: Expr_pattern : "Expr11284" Anatomy_term "WBbt:0006894" Gene "WBGene00001185" Pattern "The distal enhancer activity was observed in P6.p and its descendants from the two-cell stage and increased with time. Distal enhancer activity persisted into much later stages than the proximal enhancer did." Reference "WBPaper00005841" Reporter_gene Construct "WBConstruct00000021" Associated_feature "WBsf919543" Construct : "WBConstruct00000021" Public_name "[Distal egl-17 enhancer reporter]" Summary "[egl-17.distal::pPD122.53]" Clone "pPD122.53" //added clone back into model Sequence_feature "WBsf919543" Gene "WBGene00001185" Fusion_reporter "GFP" Construction_summary "Distal egl-17 enhancer inserted into the pPD122.53 vector." Reference "WBPaper00005841"
Expression objects described with reporter fusions that do not have a classical Ex transgene designation
Expr_pattern : "Expr11598" Anatomy_term "WBbt:0003681" Certain Anatomy_term "WBbt:0005772" Certain Anatomy_term "WBbt:0006749" Certain Gene "WBGene00001753" Life_stage "WBls:0000023" Life_stage "WBls:0000041" Pattern "gst-5 was expressed in intestine, pharynx, and circumpharyngeal neurons. Expression is seen from L1 to adult stages." Reference "WBPaper00037704" Reporter_gene Construct "WBConstruct00000021" Construct : "WBConstruct00000021" Public_name "Expr11598_Ex" Summary "[GST-5::GFP]" Driven_by_gene "WBGene00001753" Gene "WBGene00001753" Fusion_reporter "GFP" Translational_fusion Reference "WBPaper00037704"
Interaction/Regulation objects pertaining to construct
Interaction/Regulation object contains sequence feature. I am not sure shall we add construct tag in our model? --Xiaodong
Interaction : "WBInteraction000502404" Change_of_expression_level Interactor_overlapping_gene "WBGene00004077" Trans_regulator Interactor_overlapping_gene "WBGene00004077" Variation "WBVar00241166" Interactor_overlapping_gene "WBGene00009560" Trans_regulated Interactor_overlapping_gene "WBGene00009560" Expr_pattern "Expr4287" Feature_interactor "WBsf034247" Cis_regulator Interaction_summary "psa-3 expression was induced in T.p after the T cell division, and it accumulated in the posterior granddaughters. psa-3 expression in the T.p lineage was much lower in a pop-1 hypomorphic allele, q645." Reporter_gene "[psa-3::gfp], translational fusion" //This information seems like it can be captured by transgene or construct now, I am missing why this tag is needed. //Xiaodong's comments: this tag has been in the model from the beginning. it's just curator's simple notes on reporter construct. if you notice, I don't even have the precise reporter description. //We can leave as it is now, and let user have a rough idea of what reporter looks like. if they want, they can go to check construct for detailed info. //If the construct model goes in, the webteam can port the construct summary to this interaction page, likewise with the transgene summary info. But I leave it to you. Construct "WBConstruct00000021" Transcriptional Positive_regulate Anatomy_term "WBbt:0006996" Positive_regulate Anatomy_term "WBbt:0006997" Positive_regulate Anatomy_term "WBbt:0007000" Positive_regulate Anatomy_term "WBbt:0007007" Positive_regulate Anatomy_term "WBbt:0007012" Positive_regulate Anatomy_term "WBbt:0007016" Paper "WBPaper00027741"Remark "POP-1 function is known to be required in T.p to determine the neural fate. POP-1 regulates psa-3 expression in T.p through the POP-1 binding site." Construct : "WBConstruct00000021" Public_name "osEx113" Summary "[Ppsa-3::psa-3::gfp]" Construction_summary "psa-3 promoter and the entire coding region." Reporter_product "GFP" Driven_by_gene "WBGene00009560" Gene "WBGene00009560" Translational_fusion Reference "WBPaper00040473" Reference "WBPaper00027741"
