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| − | == ''' | + | == '''The Tol2kit'''== |
| − | ''Tol2kit v1. | + | ''Tol2kit v1.2, released November 2007'' |
| + | |||
| + | Generating stable transgenic zebrafish lines has historically been laborious, for three reasons: | ||
| + | * Building transgenesis constructs by conventional subcloning can be difficult, especially when using long coding or regulatory sequences | ||
| + | * Injecting plasmid DNA yields mosaic transient expression and low germline transgenesis rates | ||
| + | * When screening for stable integrations, transgenes that express fluorescent proteins (e.g. GFP) are easy to detect, but other transgenes are not. | ||
| + | |||
| + | The Tol2kit uses site-specific recombination-based cloning ([[Basic Gateway principles|multisite Gateway technology]]) to allow quick, modular assembly of promoter-coding sequence-3' tag constructs in a Tol2 transposon backbone. | ||
| + | |||
| + | It includes a variety of widely-useful [[List of entry and destination vectors|entry clones]], including ''hsp70'' and ''beta-actin'' promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and IRES-driven GFP cassettes for bicistronic expression. It includes two Tol2-based [[List of entry and destination vectors|destination vectors]], one with a ''cmlc2:egfp'' transgenesis marker. | ||
| + | |||
| + | == About this site == | ||
| + | |||
| + | This wiki provides the documentation for the Tol2kit; it is maintained by the [http://kwan-lab.org Kwan lab]. | ||
| + | |||
| + | * [[Basic Gateway principles]] | ||
| + | * [[List of entry and destination vectors|List of entry and destination clones]] | ||
| + | * [[Protocols]] | ||
| + | * [[Sample results with the Tol2kit]] | ||
| + | * [[Sources|Sources for the Tol2kit]] | ||
| + | |||
| + | If you have questions or comments about the Tol2kit, please contact Kristen Kwan <kristen.kwan@genetics.utah.edu>. If you construct other entry or destination clones, we would love to post information about them here. | ||
| + | |||
| + | == Clone distribution == | ||
| + | |||
| + | We are freely distributing all of the plasmids described here. For reasons of convenience, we usually distribute them as a complete set, spotted as DNA onto filter paper. If you have an older version (e.g. v1.0), we can send you an "upgrade kit" to the latest version. Since the destination vectors and transposase plasmid are all derived from Dr. Koichi Kawakami's original constructs, we ask that you please '''first contact Dr. Kawakami''' <kokawaka@nig.ac.jp> and execute an MTA for use of the Tol2 transposase construct and plasmids based on Tol2. | ||
| + | |||
| + | We are also happy for labs to share these plasmids or pass them on to others, '''as long as''' you pass on the address of this wiki, '''and''' the receiving lab has executed a Tol2 MTA with Dr. Kawakami. We especially encourage different labs at the same institution to share plasmids. This reduces our workload (generating maxipreps and spotting plasmid) as well as yours (growing up clones). | ||
| + | |||
| + | To request the Tol2kit, please send an email to Kristen Kwan <kristen.kwan@genetics.utah.edu>. | ||
| + | |||
| + | == Citing the Tol2kit == | ||
| + | |||
| + | A methods paper describing the Tol2kit has been published in ''Developmental Dynamics''. Here are links to the [http://www3.interscience.wiley.com/cgi-bin/abstract/116330946/ABSTRACT online version] and a [http://chien.neuro.utah.edu/pdfs/Kwan_preprint.pdf preprint]. Please cite this paper in manuscripts using the Tol2kit. | ||
== Links == | == Links == | ||
| − | [http://tol2kit.blogspot.com Tol2kit blog for discussion and questions about the system] | + | * [http://tol2kit.blogspot.com Tol2kit blog for discussion and questions about the system] |
| − | [http://lawsonlab.umassmed.edu/gateway.html Lawson lab Gateway site] | + | * [http://lawsonlab.umassmed.edu/gateway.html Lawson lab Gateway site] |
| − | [ | + | * [http://kawakami.lab.nig.ac.jp Kawakami lab site] |
| + | * [[Invitrogen manual | Invitrogen multisite Gateway manual Version D 3/7/07]] | ||
| + | |||
| + | == Revision history == | ||
| + | 03-07-10 Added details to clone description for pME-Gal4VP16. Coding sequence derives from Reinhard Koester's clones described in Koester et al. 2001; VP16 fragment is in fact VP16[413-470], a VP16 transactivation domain that has been partially truncated, reducing toxicity of the fusion protein.<br> | ||
| + | 10-12-09 Added description of clone 550 (absolutely correct version of pME-mCherryCAAX). The previous version of this clone, #450, was contaminated with the H80D mutation in some preps. <br> | ||
| + | 04-28-09 Minor edits to list of clones page; pDONR clones had all been listed as middle clones. Hat tip to Yuk Fai Leung and Jim Lister.<br> | ||
| + | 12-18-07 Added information about using ApE to [[Protocols#Assembling_sequences_for_expression_clones|predict expression clone sequences]].<br> | ||
| + | 12-18-07 Added Genbank-format sequence for all clones currently being distributed.<br> | ||
| + | 12-09-07 Minor edits to the [[att site sequences| att seq list page]] to fix a couple of sequences and clarify names (especially the attBXX_primer sequences).<br> | ||
| + | 11/5/07 Added a [[inserts and ends|page]] with instructions and sequences for assembling predicted sequence for expression clones.<br> | ||
| + | 11/2/07 Minor upgrade (to v1.2), adding middle clones for mCherryCAAX (no mutations); EGFP (no stop) and mCherry (no stop).<br> | ||
| + | 10/11/07 Added an [[Invitrogen manual | archived copy]] of the Invitrogen manual, because the previous live link broke, and because their nomenclature seems to change unpredictably.<br> | ||
| + | 9/23/07 Added the Sources page. The wiki is now substantially complete and is entering a maintenance phase. We will continue to correct errors and add information on new clones as the kit evolves.<br> | ||
| + | 9/12/07 Added a preprint version of the Tol2kit methods paper; various other minor changes.<br> | ||
| + | 8/17/07 Wiki URL: to fix a problem that some users had with firewall blocking of the wiki, we have slightly reengineered access to the site. You now must add a forward slash to the end of the URL: http://chien.neuro.utah.edu/tol2kitwiki/<br> | ||
| + | 6/7/07 List of clones: columns for version numbers and MTAs have been added.<br> | ||
| + | 6/7/07 Clone sequences: six bases (a KpnI site) were missing from our predicted sequences for pDestTol2pA, pDestTol2pA2, pDestTol2CG, and pDestTol2CG2. Maps and sequences have all been corrected.<br> | ||
| + | 6/5/07 Sample results: Added images of [[Sample results with the Tol2kit|sample results]] using IRES constructs.<br> | ||
| + | 5/11/07 Links: note that Invitrogen has issued a new version of the multisite Gateway manual, version D dated 3/7/07, which includes use of the new LR Clonase II Plus enzyme.<br> | ||
| + | 5/1/07 Main page: Moved revision history here from clone list page.<br> | ||
| + | 4/18/07 Sidebar: Added links for quick navigation.<br> | ||
| + | 4/17/07 Clone list: Added sequences for pDestTol2pA and pDestTol2CG (obsolete destination vectors).<br> | ||
| + | 3/23/07 Clone list: Sequences and maps now uploaded for all constructs.<br> | ||
| + | 2/20/07 Clone list: Sizes in bp have been added for all constructs.<br> | ||
== Wiki help == | == Wiki help == | ||
Latest revision as of 05:30, 20 May 2022
Contents
The Tol2kit
Tol2kit v1.2, released November 2007
Generating stable transgenic zebrafish lines has historically been laborious, for three reasons:
- Building transgenesis constructs by conventional subcloning can be difficult, especially when using long coding or regulatory sequences
- Injecting plasmid DNA yields mosaic transient expression and low germline transgenesis rates
- When screening for stable integrations, transgenes that express fluorescent proteins (e.g. GFP) are easy to detect, but other transgenes are not.
The Tol2kit uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of promoter-coding sequence-3' tag constructs in a Tol2 transposon backbone.
It includes a variety of widely-useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and IRES-driven GFP cassettes for bicistronic expression. It includes two Tol2-based destination vectors, one with a cmlc2:egfp transgenesis marker.
About this site
This wiki provides the documentation for the Tol2kit; it is maintained by the Kwan lab.
- Basic Gateway principles
- List of entry and destination clones
- Protocols
- Sample results with the Tol2kit
- Sources for the Tol2kit
If you have questions or comments about the Tol2kit, please contact Kristen Kwan <kristen.kwan@genetics.utah.edu>. If you construct other entry or destination clones, we would love to post information about them here.
Clone distribution
We are freely distributing all of the plasmids described here. For reasons of convenience, we usually distribute them as a complete set, spotted as DNA onto filter paper. If you have an older version (e.g. v1.0), we can send you an "upgrade kit" to the latest version. Since the destination vectors and transposase plasmid are all derived from Dr. Koichi Kawakami's original constructs, we ask that you please first contact Dr. Kawakami <kokawaka@nig.ac.jp> and execute an MTA for use of the Tol2 transposase construct and plasmids based on Tol2.
We are also happy for labs to share these plasmids or pass them on to others, as long as you pass on the address of this wiki, and the receiving lab has executed a Tol2 MTA with Dr. Kawakami. We especially encourage different labs at the same institution to share plasmids. This reduces our workload (generating maxipreps and spotting plasmid) as well as yours (growing up clones).
To request the Tol2kit, please send an email to Kristen Kwan <kristen.kwan@genetics.utah.edu>.
Citing the Tol2kit
A methods paper describing the Tol2kit has been published in Developmental Dynamics. Here are links to the online version and a preprint. Please cite this paper in manuscripts using the Tol2kit.
Links
- Tol2kit blog for discussion and questions about the system
- Lawson lab Gateway site
- Kawakami lab site
- Invitrogen multisite Gateway manual Version D 3/7/07
Revision history
03-07-10 Added details to clone description for pME-Gal4VP16. Coding sequence derives from Reinhard Koester's clones described in Koester et al. 2001; VP16 fragment is in fact VP16[413-470], a VP16 transactivation domain that has been partially truncated, reducing toxicity of the fusion protein.
10-12-09 Added description of clone 550 (absolutely correct version of pME-mCherryCAAX). The previous version of this clone, #450, was contaminated with the H80D mutation in some preps.
04-28-09 Minor edits to list of clones page; pDONR clones had all been listed as middle clones. Hat tip to Yuk Fai Leung and Jim Lister.
12-18-07 Added information about using ApE to predict expression clone sequences.
12-18-07 Added Genbank-format sequence for all clones currently being distributed.
12-09-07 Minor edits to the att seq list page to fix a couple of sequences and clarify names (especially the attBXX_primer sequences).
11/5/07 Added a page with instructions and sequences for assembling predicted sequence for expression clones.
11/2/07 Minor upgrade (to v1.2), adding middle clones for mCherryCAAX (no mutations); EGFP (no stop) and mCherry (no stop).
10/11/07 Added an archived copy of the Invitrogen manual, because the previous live link broke, and because their nomenclature seems to change unpredictably.
9/23/07 Added the Sources page. The wiki is now substantially complete and is entering a maintenance phase. We will continue to correct errors and add information on new clones as the kit evolves.
9/12/07 Added a preprint version of the Tol2kit methods paper; various other minor changes.
8/17/07 Wiki URL: to fix a problem that some users had with firewall blocking of the wiki, we have slightly reengineered access to the site. You now must add a forward slash to the end of the URL: http://chien.neuro.utah.edu/tol2kitwiki/
6/7/07 List of clones: columns for version numbers and MTAs have been added.
6/7/07 Clone sequences: six bases (a KpnI site) were missing from our predicted sequences for pDestTol2pA, pDestTol2pA2, pDestTol2CG, and pDestTol2CG2. Maps and sequences have all been corrected.
6/5/07 Sample results: Added images of sample results using IRES constructs.
5/11/07 Links: note that Invitrogen has issued a new version of the multisite Gateway manual, version D dated 3/7/07, which includes use of the new LR Clonase II Plus enzyme.
5/1/07 Main page: Moved revision history here from clone list page.
4/18/07 Sidebar: Added links for quick navigation.
4/17/07 Clone list: Added sequences for pDestTol2pA and pDestTol2CG (obsolete destination vectors).
3/23/07 Clone list: Sequences and maps now uploaded for all constructs.
2/20/07 Clone list: Sizes in bp have been added for all constructs.
Wiki help
Consult the User's Guide for information on using the wiki software.