{"id":749,"date":"2014-01-31T19:22:58","date_gmt":"2014-02-01T01:22:58","guid":{"rendered":"http:\/\/wormbuilder.org\/old\/?page_id=749"},"modified":"2020-02-15T22:17:25","modified_gmt":"2020-02-16T04:17:25","slug":"land","status":"publish","type":"page","link":"https:\/\/wormbuilder.org\/old\/","title":{"rendered":""},"content":{"rendered":"<h4 style=\"text-align: center;\">Recent news<\/h4>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Wormbook chapter on C. elegans transgenic methods is out in Genetics.\u00a0<\/strong><\/p>\n<p><em>Aug 23, 2019<\/em><\/p>\n<p><a href=\"https:\/\/www.genetics.org\/content\/212\/4\/959\"><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1560\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1560\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics.png\" data-orig-size=\"1144,1494\" data-comments-opened=\"0\" data-image-title=\"Genetics\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-784x1024.png\" class=\"alignleft size-medium wp-image-1560\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-230x300.png\" alt=\"\" width=\"230\" height=\"300\" srcset=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-230x300.png 230w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-768x1003.png 768w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-784x1024.png 784w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-100x131.png 100w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-150x196.png 150w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-200x261.png 200w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-300x392.png 300w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-450x588.png 450w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-600x784.png 600w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics-900x1175.png 900w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2020\/02\/Genetics.png 1144w\" sizes=\"auto, (max-width: 230px) 100vw, 230px\" \/><\/a><\/p>\n<p>My review \/ Wormbook chapter with <a href=\"http:\/\/nancelab.med.nyu.edu\/\">Jeremy Nance (NYU)<\/a> is\u00a0out now in Genetics (paper <a href=\"https:\/\/www.genetics.org\/content\/212\/4\/959\">here<\/a>). Please email me if you don&#8217;t have access to Genetics and I will send you a pdf.<\/p>\n<p>We try to cover the rapidly expanding set of tools that have become developed for <em>C. elegans<\/em> with a focus on recent developments. However, we&#8217;ve also tried to summarize some of the accumulated &#8220;wisdom&#8221; from many different labs to improve the chance of generating a functional transgene.<\/p>\n<p>The different sections cover:<\/p>\n<ul>\n<li><strong><em>C. elegans<\/em> Genes and Transgene Structures<\/strong><\/li>\n<li><strong>Multicopy Transgenesis<\/strong><\/li>\n<li><strong>Integrating arrays<\/strong><\/li>\n<li><strong>Transposon-Mediated Genetic Engineering<\/strong><\/li>\n<li><strong>CRISPR\/Cas9-Mediated Transgenesis<\/strong><\/li>\n<li><strong>Conditional Gene and Protein Inactivation<\/strong><\/li>\n<li><strong>Bipartite Systems for Temporal and Spatial Control of Expression<\/strong><\/li>\n<\/ul>\n<p>We hope the review will be a useful &#8220;entry point&#8221; for researchers that are new to <em>C. elegans<\/em> while also having information that for the seasoned <em>C. elegans<\/em> scientist.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Paper on non-Mendelian inheritance now published at Developmental Cell.<\/strong><\/p>\n<p><em>Feb 25, 2019<\/em><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1373\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1373\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract.jpg\" data-orig-size=\"375,375\" data-comments-opened=\"0\" data-image-meta=\"{&quot;title&quot;:&quot;Graphical_abstract_121918&quot;,&quot;orientation&quot;:&quot;1&quot;}\" data-image-title=\"Graphical_abstract_121918\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract.jpg\" class=\"aligncenter size-full wp-image-1373\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract.jpg\" alt=\"\" width=\"375\" height=\"375\" srcset=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract.jpg 375w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract-150x150.jpg 150w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract-300x300.jpg 300w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract-100x100.jpg 100w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/graphic-abstract-200x200.jpg 200w\" sizes=\"auto, (max-width: 375px) 100vw, 375px\" \/><\/p>\n<p>Karen&#8217;s paper on non-Mendelian inheritance is now online and can be found <a href=\"https:\/\/www.cell.com\/developmental-cell\/fulltext\/S1534-5807(19)30048-6\">here<\/a>. Please send me a request if you have trouble accessing the paper. In the paper, we generate a set of strains (available at <a href=\"https:\/\/cgc.umn.edu\/strain\/search?st1=ccTi1594&amp;sf1=all&amp;xt1=&amp;xf1=all&amp;st2=&amp;sf2=strain&amp;xt2=&amp;xf2=strain&amp;st3=&amp;sf3=genotype&amp;xt3=&amp;xf3=genotype&amp;st4=&amp;sf4=species&amp;xt4=&amp;xf4=species\">cgc<\/a>) that facilitate crosses where segregation follows a non-Mendelian pattern by overexpressing GPR-1, as shown by <a href=\"https:\/\/www.nature.com\/articles\/nbt.3643\">Besseling and Bringmann<\/a> (2016).\u00a0 We demonstrate that the strains can be used to &#8220;transplant&#8221; mitochondria between strains and show lineage-specific requirements for vulval induction.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1376\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1376\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/Red-green-pharynx-full.gif\" data-orig-size=\"595,510\" data-comments-opened=\"0\" data-image-title=\"Red-green-pharynx-full\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/Red-green-pharynx-full.gif\" class=\"aligncenter size-full wp-image-1376\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/Red-green-pharynx-full.gif\" alt=\"\" width=\"595\" height=\"510\" \/><\/p>\n<p>Other than making really cool pharyngeal expression patterns, the strains should be useful for determining maternal and paternal contributions to development and epigenetic, transgenerational inheritance.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>The lab is growing!<\/strong><\/p>\n<p><em>Feb 25, 201<\/em><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1368\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1368\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/lab_outside_Feb_2019.gif\" data-orig-size=\"612,792\" data-comments-opened=\"0\" data-image-title=\"lab_outside_Feb_2019\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/lab_outside_Feb_2019.gif\" class=\"aligncenter size-full wp-image-1368\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2019\/02\/lab_outside_Feb_2019.gif\" alt=\"\" width=\"612\" height=\"792\" \/><\/p>\n<p>After a bit more than a year at KAUST, the lab now has two postdocs (Sonia el Mouridi and Amhed Velazquez), two Ph.D. students (Monika Priyardishini and Mohammed Aljohani), and one visiting student (Alhanouf).<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>New position as Assistant Professor at KAUST<\/strong><\/p>\n<p><em>Jan 2, 2018<\/em><\/p>\n<p><a href=\"https:\/\/syngenbio.kaust.edu.sa\/Pages\/Home.aspx\"><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1281\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1281\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small.png\" data-orig-size=\"630,243\" data-comments-opened=\"0\" data-image-title=\"KAUST_wide_exterior_feature_small\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small.png\" class=\"aligncenter wp-image-1281 size-full\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small.png\" alt=\"KAUST library at night\" width=\"630\" height=\"243\" srcset=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small.png 630w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-300x116.png 300w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-100x39.png 100w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-150x58.png 150w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-200x77.png 200w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-450x174.png 450w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/KAUST_wide_exterior_feature_small-600x231.png 600w\" sizes=\"auto, (max-width: 630px) 100vw, 630px\" \/><\/a><\/p>\n<p>I am excited to have recently started my own <a href=\"https:\/\/syngenbio.kaust.edu.sa\/Pages\/Home.aspx\">research group<\/a> at <a href=\"https:\/\/en.wikipedia.org\/wiki\/King_Abdullah_University_of_Science_and_Technology\">KAUST<\/a> (King Abdullah University of Science &amp; Technology) in Saudi Arabia. KAUST is a very modern, international university with over 100 nationalities represented on campus, approximately 40% female students, and a focus on Engineering &amp; Science. The university is near <a href=\"https:\/\/www.google.com.sa\/maps\/place\/King+Abdullah+University+of+Science+and+Technology\/@26.8142244,32.0042515,5.13z\/data=!4m5!3m4!1s0x15c11c505768a2f1:0x36139c007214e6bc!8m2!3d22.309465!4d39.104697\">Jeddah<\/a> on the coast of the Red Sea.<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1276\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1276\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea.jpg\" data-orig-size=\"976,395\" data-comments-opened=\"0\" data-image-title=\"KAUST Red Sea\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea.jpg\" class=\"aligncenter size-full wp-image-1276\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea.jpg\" alt=\"The beacon is KAUST's symbol\" width=\"976\" height=\"395\" srcset=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea.jpg 976w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-300x121.jpg 300w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-768x311.jpg 768w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-100x40.jpg 100w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-150x61.jpg 150w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-200x81.jpg 200w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-450x182.jpg 450w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-600x243.jpg 600w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2018\/01\/beacon-sea-900x364.jpg 900w\" sizes=\"auto, (max-width: 976px) 100vw, 976px\" \/><\/p>\n<p>Although I realize Saudi Arabia is not the most obvious choice for establishing a laboratory, I was very attracted by the scientific community, the resources available for research, and about being part of &#8220;building&#8221; the academic environment at a young, ambitious university in a region, that is eager to educate their youth.<\/p>\n<p><strong>The laboratory is actively looking for postdocs<\/strong>, so if you are interested in developing large-scale genome engineering technologies with a focus on genome organization, then I encourage you to apply <a href=\"https:\/\/syngenbio.kaust.edu.sa\/Pages\/JoinUs.aspx\">here<\/a>.<\/p>\n<p>The university has very attractive <a href=\"http:\/\/studentopportunities.kaust.edu.sa\/\">programs<\/a> with all expenses paid to encourage undergraduate visitors. There is a poster presentation at the annual Winter Enrichment Program (this year, <a href=\"https:\/\/enrichment.kaust.edu.sa\/pages\/home\">Human Machine Future<\/a>). Also, there are month-long research projects with a stipend offered via the <a href=\"https:\/\/vsrp.kaust.edu.sa\/Pages\/Home.aspx\">Visiting Student Research Program<\/a>\u00a0where we have <a href=\"https:\/\/vsrp.kaust.edu.sa\/Pages\/Synthetic%20biology%20achieving%20high%20efficiency%20genome%20editing%20in%20a%20multicellular%20organism.aspx\">a project<\/a> listed. I strongly encourage an application; it is a great scientific and cultural experience, with some fun extracurricular activities thrown in together with the cohort of students (snorkeling and diving are spectacular here). So far, one female undergraduate student from a <em>C. elegans<\/em> lab in the US was &#8220;brave&#8221; enough to apply; she&#8217;ll come for WEP this January.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>New Reagents Deposited with Addgene<\/strong><\/p>\n<p><em>Feb\u00a08, 2017<\/em><\/p>\n<p>We have deposited several plasmids with C. elegans optimized fluorophores with and without PATC. In our hands, including PATCs significantly diminishes epigenetic silencing in the germline but has no obvious positive or negative effect on somatic expression. The plasmids are described <a href=\"http:\/\/wormbuilder.org\/old\/reagents\/codon-optimized-flourophores\/\">here<\/a> and can be accessed at Addgene <a href=\"https:\/\/www.addgene.org\/browse\/article\/22567\/\">here<\/a>.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Paper on Periodic An\/Tn Clusters (PATCs) published<\/strong><\/p>\n<p><em>July 6, 2016<\/em><\/p>\n<p>Our paper on epigenetic silencing of transgenes in the <em>C. elegans<\/em> germline just came <a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/27374334\">out<\/a>.<\/p>\n<p><a href=\"http:\/\/www.cell.com\/cell\/fulltext\/S0092-8674(16)30676-6\"><img loading=\"lazy\" decoding=\"async\" data-attachment-id=\"1206\" data-permalink=\"https:\/\/wormbuilder.org\/old\/?attachment_id=1206\" data-orig-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2.png\" data-orig-size=\"558,555\" data-comments-opened=\"0\" data-image-title=\"Graphic Abstract\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2.png\" class=\"aligncenter wp-image-1206 size-full\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2.png\" alt=\"An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.\" width=\"558\" height=\"555\" srcset=\"https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2.png 558w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2-150x149.png 150w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2-300x298.png 300w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2-100x99.png 100w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2-200x199.png 200w, https:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2014\/01\/graphic_abstract_2-450x448.png 450w\" sizes=\"auto, (max-width: 558px) 100vw, 558px\" \/><\/a><\/p>\n<ul>\n<li>Periodic, non-coding DNA can prevent transgenes from stochastic silencing in germline<\/li>\n<li>Non-coding content of genes is shaped by genomic context and heterochromatin domains<\/li>\n<li>Conditioning of active DNA may allow cells to distinguish foreign from host genes<\/li>\n<\/ul>\n<p>The worm genome contains a prevalent (~10% of the genomic sequence) non-coding DNA feature called PATCs, which is highly enriched &#8220;around&#8221; genes expressed in the germline. PATCs are highly enriched in genes that reside in repressive chromatin environments, suggesting that this non-coding protects\u00a0endogenous genes from epigenetic silencing.\u00a0We propose\u00a0that PATCs may also play a role in a\u00a0basic cellular immune system to detect foreign DNA. In this model, germ cells use this abundant non-coding DNA as a signature to identify endogenous genes and to silence foreign DNA (transposons, retroviruses, transgenes) that lack PATCs by default. In line with this, we find that transgenes with PATCs are much less frequently silenced in the\u00a0germline from permissive and repressive chromatin domains.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Fluorescent markers strains with GFP insertions.\u00a0<\/strong><\/p>\n<p>In collaboration with Ann Rougvie at the CGC, we have generated a set of strains carrying GFP markers at defined genomic locations. The strains are available from the CGC and you can search for the strains based on chromosome, fluorescent marker, and the selection marker (see <a title=\"Fluorescent marker strains\" href=\"http:\/\/wormbuilder.org\/old\/reagents\/fluorescent-marker-strains\/\">Fluorescent marker strains<\/a>). The fluorescence is visible on a fluorescence dissection microscope and the strains can be used to build\u00a0\u00a0double mutants in crosses or for outcrossing mutations isolated in genetic screens. The strains complement the previously published set of strains with insertions of tdTomato and brings the total to 294 strains.<\/p>\n<p>In addition, the CGC is generating fluorescently marked versions of some of the classic balancer strains by targeted integration using Cas9. We have generated a marked MT1000 strain (unc-5(e53)\/nT1 IV; dpy-11(e224)\/nT1 V) by inserting Peft-3:GFP into Chr. V at 2.8 MB. The strain can be used as a balancer even though the cytosolic GFP is somewhat dim. We have therefore switched to inserting a brighter Peft-3:tdTomato(NLS) marker.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Move to Andrew Fire&#8217;s lab at Stanford<\/strong><\/p>\n<p><em>Sept 16, 2014<\/em><\/p>\n<p>I have just recently moved to <a href=\"http:\/\/firelab.stanford.edu\/\" target=\"_blank\" rel=\"noopener\">Andrew Fire<\/a>&#8216;s lab at Stanford! In addition to developing techniques to engineer <em>C. elegans<\/em>, I will also work on understanding how cells in the germline identify foreign and endogenous genes as a postdoc in Andy&#8217;s lab (technically, a &#8220;visiting instructor&#8221;).<\/p>\n<p>Thanks to <a href=\"http:\/\/biologylabs.utah.edu\/jorgensen\/Labsite\/Individual%20Pages\/IndividualsJorgensen.html\" target=\"_blank\" rel=\"noopener\">Erik<\/a>\u00a0and everyone in the lab for a fantastic time at University of Utah!<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Antibiotic selection protocol<\/strong><\/p>\n<p><em>July\u00a02, 2014<\/em><\/p>\n<p>I have added a <a title=\"Antibiotic selection\" href=\"http:\/\/wormbuilder.org\/old\/reagents\/antibiotic-selection\/\">page<\/a> with the protocol we use in Erik&#8217;s lab for antibiotic selection.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<p><strong>Variable efficiency of <em>peel-1<\/em> negative selection<\/strong><\/p>\n<p><em>June\u00a017, 2014<\/em><\/p>\n<p>For no\u00a0obvious reason, I have recently observed large variability in the efficiency in using the <em>peel-1<\/em> negative selection to kill animals with extra-chromosomal arrays. When I initially characterized the negative selection (Fr\u00f8kj\u00e6r-Jensen <em>et al.<\/em>, 2010) it was very efficient at killing essentially all animals with extra-chromosomal arrays. For a long time, I and others in Erik&#8217;s lab picked animals with mosSCI or miniMos insertions based on surviving the heat-shock and found essentially no false positives (by a secondary screen for mCherry co-injection markers). However for the past two summers, the negative selection has become very inefficient even when using the same injection mixes that previously worked. It is very strange but it coincides closely with the installation of a new evaporative cooling system in the building and the summer months. I have performed a number of experiments to enhance the efficiency of negative selection:<\/p>\n<p>&#8211; Using the genomic <em>peel-1<\/em> fragment instead of cDNA.<\/p>\n<p>&#8211; Heat-shock in water bath instead of in an air incubator.<\/p>\n<p>&#8211; Higher temperature heat-shock (1 hour at 37\u00b0C instead of 34\u00b0C).<\/p>\n<p>&#8211; Doubling the concentration of the <em>peel-1<\/em> plasmid (pMA122)<\/p>\n<p><em>None of these changes improved the negative selection.<\/em><\/p>\n<p>I apologize if others are observing similar variability. This was <strong>not<\/strong> something I observed when originally characterizing the negative selection. And if anyone has figured out how to reduce the variability (other than wait for fall and winter) then please send me an email. Or write up a paper on how &#8220;A sperm-delivered toxin can determine the seasons in the short-lived model organism <em>C. elegans<\/em>&#8220;&#8230;<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><\/div>\n<div class=\"su-spoiler su-spoiler-style-default su-spoiler-icon-plus su-spoiler-closed\" data-scroll-offset=\"0\" data-anchor-in-url=\"no\"><div class=\"su-spoiler-title\" tabindex=\"0\" role=\"button\"><span class=\"su-spoiler-icon\"><\/span>Old news...<\/div><div class=\"su-spoiler-content su-u-clearfix su-u-trim\">\n<p><strong>Possible issues with the universal insertion site on LGI<\/strong><\/p>\n<p><em>March 18, 2014<\/em><\/p>\n<p>I have heard from a few people using the universal insertion site on LGI (<em>oxTi185<\/em><em>,\u00a0<a href=\"http:\/\/www.cgc.cbs.umn.edu\/strain.php?id=24777\" target=\"_blank\" rel=\"noopener\">EG8078<\/a>)\u00a0<\/em>that insertions may be more difficult into this locus &#8211; and that the PCR to verify the insertion site gives aberrant bands (mos1 band + wt band). I am trying to figure out if there is a problem with the deposited strain or if something else is wrong. For now, I would preferably use one of the other insertion sites. Sorry for the inconvenience.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p><strong>miniMos and universal insertion site paper published<\/strong><\/p>\n<p><em>March 16, 2014<\/em><\/p>\n<p>The paper describing the miniMos transposon and the universal mosSCI insertion sites was just published and can be found <a href=\"http:\/\/www.nature.com\/nmeth\/journal\/vaop\/ncurrent\/full\/nmeth.2889.html\" target=\"_blank\" rel=\"noopener\">here<\/a>.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p><strong>miniMos vectors with hygromycin selection<\/strong><\/p>\n<p><em>March 2, 2014<\/em><\/p>\n<p>I have generated a set of miniMos vectors with the hygromycin selection cassette (recently developed by Jason Chin&#8217;s lab, <a href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/24130756\" target=\"_blank\" rel=\"noopener\">pubmed<\/a>). The antibiotic selections NeoR, PuroR and HygroR all work equally well in my hands and result in insertions at similar frequencies to using <em>unc-119<\/em> selection.<\/p>\n<p>The plasmids are available from Addgene and can be found <a title=\"miniMos reagents\" href=\"http:\/\/wormbuilder.org\/old\/minimos\/plasmids-2\/\">here<\/a>.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p style=\"text-align: left;\"><strong>Fluorescent mapping strains<\/strong><\/p>\n<p><span style=\"color: #999999;\"><em>Janu<\/em><em>ary<\/em><em> 31, 2014<\/em><\/span><\/p>\n<p style=\"padding-left: 30px;\">A graduate student in Erik\u2019s lab (Matt LaBella) has built two mapping strains based on fluorescent miniMos insertions. Each strain contains three distinct fluorescent markers on separate chromosomes in a\u00a0<em>him<\/em>\u00a0background. He and others in the lab has used them successfully to map new mutations to a chromosome before whole-genome sequencing. We\u2019ve deposited the mapping strains\u00a0<a title=\"EG8040\" href=\"http:\/\/www.cgc.cbs.umn.edu\/strain.php?id=25234\" target=\"_blank\" rel=\"noopener\">EG8040<\/a>\u00a0and\u00a0<a title=\"EG8041\" href=\"http:\/\/www.cgc.cbs.umn.edu\/strain.php?id=25235\" target=\"_blank\" rel=\"noopener\">EG8041<\/a>\u00a0at the CGC if you want to give them a try.<\/p>\n<p style=\"padding-left: 30px;\">See\u00a0<a title=\"Fluorescent mapping strains\" href=\"http:\/\/wormbuilder.org\/old\/fluorescent-mapping-strains\/\">here<\/a>\u00a0for more information about the markers.<\/p>\n<p style=\"padding-left: 30px;\"><div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<div id=\"post-717\">\n<p><strong>Verification of insertions into universal mosSCI sites<\/strong><\/p>\n<div><span style=\"color: #999999;\"><em>October 18, 2013<\/em><\/span><\/div>\n<div>\n<p style=\"padding-left: 30px;\">I\u2019ve had a few people ask me for sequences surrounding the universal mosSCI sites and oligos that can be used to verify insertions and homozygosity of the inserts. I have posted ApE files with the local genomic environment and the oligos we use to verify the position and homozygosity of inserts\u00a0<a title=\"Universal insert verification\" href=\"http:\/\/wormbuilder.org\/old\/universal-mossci-sites\/universal-insert-verification\/\">here<\/a>.<\/p>\n<p style=\"padding-left: 30px;\"><div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p><strong>DNA quality and MosSCI insertion frequency<\/strong><\/p>\n<\/div>\n<\/div>\n<div id=\"post-694\">\n<div><span style=\"color: #999999;\"><em>September 12, 2013<\/em><\/span><\/div>\n<div>\n<p style=\"padding-left: 30px;\">Morris Maduro has previously pointed out that the quality of the DNA preparation for injection made a large difference in the number of rescued F1 progeny (see\u00a0<a title=\"Worm Breeders Gazette\" href=\"http:\/\/www.wormbook.org\/wbg\/articles\/volume-19-number-1\/omission-of-rnase-and-addition-of-ethanol-precipitation-improve-transgenesis-using-spin-column-purified-dna\/\" target=\"_blank\" rel=\"noopener\">Worm Breeders Gazette<\/a>). Since we had started seeing some variability in the insertion frequency, I tested different mini and midiprep kits for toxicity and insertion frequency.<\/p>\n<p style=\"padding-left: 30px;\">I tested three kits:<\/p>\n<ul>\n<li>Qiaprep Spin miniprep kit (cat. no. 27106) (with RNAse and no EtOh precipitation)<\/li>\n<li>Qiagen Plasmid Plus Midi kit (cat. no. 12943)<\/li>\n<li>Invitrogen PureLink HQ Mini Plasmid Purification Kit (cat. no. K2100-01) (Mihail Sarov had pointed out that this kit is very efficient in his hands)<\/li>\n<\/ul>\n<p>Experiment: I injected a 4.6 kb transgene (Peft-3:GFP:tbb-2 cb-unc-119) for insertion at the ttTi5605 site. I prepared all the DNA for the injection mix with each kit including the co-injection markers. \u00a0(pCFJ601, pMA122, pGH8, pCFJ90, pCFJ104). I did three sets of injections of 20 animals for each construct.<\/p>\n<p>Here are the results:<\/p>\n<p><img loading=\"lazy\" decoding=\"async\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2013\/09\/F1sData-3.png\" alt=\"Number of F1s\" width=\"284\" height=\"314\" \/><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" src=\"http:\/\/wormbuilder.org\/old\/wp-content\/uploads\/2013\/09\/MosSCI-insertion-frequency.png\" alt=\"MosSCI insertion frequency\" width=\"287\" height=\"314\" \/><\/p>\n<p>Data shown is mean +- SEM.<\/p>\n<p>As you can see, the Invitrogen kit performed best in terms of F1 animals and insertion frequency. The midikit also did well, whereas the miniprep kit from Qiagen clearly gave reduced number of F1 progeny and lower insertion frequency.<\/p>\n<p>So, although the Invitrogen is a little spendy, it may be worth it for making plasmids for injection.<\/p>\n<p style=\"padding-left: 30px;\"><\/div><\/div>\n<p style=\"padding-left: 30px;\"><div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<div id=\"post-686\">\n<p><strong>Problems with pCFJ1324 and pCFJ1319 LacO vectors from Addgene kit<\/strong><\/p>\n<div>\n<p><em>updated 10-12-2013<\/em><\/p>\n<p>I have re-transformed the plasmids into\u00a0<a title=\"EPI400\" href=\"http:\/\/www.epibio.com\/applications\/genomic-library-construction-kits-gene-pcr-cloning-kits\/cloning-cdna\/copycutter-epi400-electrocompetent-e-coli?details\" target=\"_blank\" rel=\"noopener\">EPI400<\/a>\u00a0cells and verified by digest that the LacO repeats are intact in the original version of the plasmids. The plasmid was resubmitted to Addgene 9-26-2013 and the fully intact plasmid should be available for distribution shortly.<\/p>\n<p><em>8-21-2013<\/em><\/p>\n<p>The vectors pCFJ1324 and pCFJ1319\u00a0in the miniMos kit contains a 256x LacO repeat. When we digested the vector it was the correct size but the repeats are prone to deletion when propagated in bacteria. Mike Nonet has digested several colonies from the kit distributed by Addgene and it appears that the bacterial clones being distributed have almost no LacO repeats.<\/p>\n<p>I will re-transform the vectors into EPI400 bacteria, that keep plasmids at low copy number until induced with arabinose. If that fixes the problem, I will deposit the bacterial strain again.<\/p>\n<p>Sorry for the inconvenience.<\/p>\n<\/div>\n<\/div>\n<div id=\"post-675\">\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p><strong>miniMos reagents available from Addgene<\/strong><\/p>\n<div><em>July 30, 2013<\/em><\/div>\n<div>\n<p>We have released the miniMos reagents prior to publication so that other researchers can use them without having to wait.<\/p>\n<p>The plasmids can be ordered either individually (see\u00a0<a title=\"Plasmids\" href=\"http:\/\/wormbuilder.org\/old\/minimos\/plasmids-2\/\">plasmids<\/a>) or as a kit (29 plasmids \u2013 8 co-injection vectors, 16 cloning vectors, 3 positive controls and 2 LacO vectors) from Addgene. Please see\u00a0<a title=\"this page\" href=\"http:\/\/www.addgene.org\/minimos\/\" target=\"_blank\" rel=\"noopener\">this page<\/a>\u00a0for information about ordering the kit. If you plan to order more than 7 plasmids it is cheaper to order the full kit. Addgene is a non-profit plasmid repository and we do not receive any compensation or commission \u2013 it is just the easiest way to distribute plasmids and saves us a lot of time dealing with shipping and MTAs.<\/p>\n<p>Also, several people in the lab are in the process of developing a gene-trap system based on the miniMos transposon. Please don\u2019t use the miniMos reagents prior to publication to compete with these efforts. We will release the gene-trap reagents as soon as possible.<\/p>\n<p>Please let me know if you have any problems with the reagents \u2013 or if you notice mistakes in the protocols or plasmid files \u2013 so I can fix them as soon as possible. Using the\u00a0<a title=\"Feedback\" href=\"http:\/\/wormbuilder.org\/old\/feedback\/\" target=\"_blank\" rel=\"noopener\">feedback<\/a>\u00a0option is easiest.<\/p>\n<p>Please cite \u201cC. Fr\u00f8kj\u00e6r-Jensen and E.M. Jorgensen, unpublished reagents\u201d if you manage to use the reagents in a publication before we publish the method.<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<\/div>\n<\/div>\n<div id=\"post-581\">\n<p><strong>miniMos reagents<\/strong><\/p>\n<div><em>July 12, 2013<\/em><\/div>\n<div>\n<p>We are planning to release the miniMos reagents through Addgene. They\u2019ve promised us that they should be ready for distrubtion by the end of July. I\u2019ll post information here \u2013 as well as detailed protocols for insertion and for mapping insertions \u2013 as soon as the reagents are available from Addgene.<\/p>\n<p>&#8211; Christian<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<p><strong>New site<\/strong><\/p>\n<\/div>\n<\/div>\n<div id=\"post-1\">\n<div><em>June 21, 2013<\/em><\/div>\n<div>\n<p>Hello and welcome!<\/p>\n<p>I decided to update the old site describing mos1-mediated techniques to modify the\u00a0<em>C.elegans<\/em>\u00a0genome. I developed the techniques together with others in the\u00a0<a href=\"http:\/\/biologylabs.utah.edu\/jorgensen\/\" target=\"_blank\" rel=\"noopener\">Jorgensen laboratory<\/a>\u00a0(mainly\u00a0<a href=\"http:\/\/biologylabs.utah.edu\/jorgensen\/Labsite\/Individual%20Pages\/IndividualsDavis.html\" target=\"_blank\" rel=\"noopener\">Wayne Davis<\/a>) and several collaborators outside the lab. I hope the protocols, links to reagents and strains will facilitate the use of the techniques for whatever you are interested in using them for. Please send me a note under the\u00a0<a title=\"Feedback\" href=\"http:\/\/wormbuilder.org\/old\/feedback\/\" target=\"_blank\" rel=\"noopener\">Feedback<\/a>\u00a0link if you have specific questions that are not answered here \u2013 or notice any problems with reagents or strains.<\/p>\n<p>&#8211; Christian<\/p>\n<div class=\"su-divider su-divider-style-default\" style=\"margin:15px 0;border-width:3px;border-color:#999999\"><a href=\"#\" style=\"color:#999999\">Top<\/a><\/div>\n<\/div>\n<\/div>\n[\/su_spoiler]\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Recent news Wormbook chapter on C. elegans transgenic methods is out in Genetics.\u00a0 Aug 23, 2019 &hellip;<\/p>\n<p class=\"read-more\"> <a class=\"more-link\" href=\"https:\/\/wormbuilder.org\/old\/\">  Read more \u2192<\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"parent":32,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"full-width-page.php","meta":{"footnotes":""},"class_list":["post-749","page","type-page","status-publish","hentry"],"jetpack_shortlink":"https:\/\/wp.me\/P3eUbV-c5","jetpack_sharing_enabled":false,"_links":{"self":[{"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/pages\/749","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=749"}],"version-history":[{"count":9,"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/pages\/749\/revisions"}],"predecessor-version":[{"id":1563,"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/pages\/749\/revisions\/1563"}],"up":[{"embeddable":true,"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=\/wp\/v2\/pages\/32"}],"wp:attachment":[{"href":"https:\/\/wormbuilder.org\/old\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=749"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}