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Cloning Mammalian Cells with the Agarose Method

Posted by Guest Blogger on Sep 7, 2017 8:17:41 AM

This post was contributed by guest blogger Iris Lindberg, Professor at the University of Maryland School of Medicine.

In the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings to isolate colonies of antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning rings, the remainder of the colonies on the plate dried out and died. The alternative to cloning rings, dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since only wells with one cell can give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require extra serum to survive at low densities - adding to the expense of dilution cloning.

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Topics: Lab Tips, Techniques

Pushing the Limits of DNA Assembly

Posted by Guest Blogger on Sep 6, 2017 9:01:33 AM

This post was contributed by guest blogger Lydia Morrison from New England Biolabs.

What is DNA assembly? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA assembly available, including: Gibson Assembly®, BioBrick® Assembly, Golden Gate Assembly, and NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using homologous regions to guide the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of Type IIS restriction enzymes results in scarless assembly. Finally, the NEBuilder® HiFi DNA Assembly method from New England Biolabs® has minimal upfront requirements and allows you to expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments, and generate constructs for producing single-guide RNAs – but it also allows you to skip purification steps and end-repair steps with well-designed fragment overlap sections, while still creating scarless plasmid inserts. 

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Topics: Techniques, Plasmid Cloning

3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

Posted by Guest Blogger on Sep 5, 2017 9:58:42 AM

This post was contributed by guest bloggers Dominik Paquet and Dylan Kwart from Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in NYC.

The CRISPR/Cas9 system is a versatile tool for precise gene editing in many organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred to as ‘indels’. While this is convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence changes.

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Topics: CRISPR

Plasmids 101: Monitoring Cell Mobility Using Fluorescent Proteins

Posted by Benoit Giquel on Aug 15, 2017 9:24:39 AM

In complex metazoans, rapid cell division and large scale cell mobility are essential processes during embryonic development. These are required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated body. In contrast, these dynamic processes are largely absent in adult organisms, where tissues structures are more stable and local movements predominate (e.g. a basal progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale mobility with movement from the bone marrow and other lymphoid organs to specific tissues where they can scan for any signs of danger. In this post we’ll focus on how fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques used here could be adapted to studying other systems in which there is large scale cellular movement throughout an organism.

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Topics: Plasmids 101, Fluorescent Proteins

Human Germline Editing Using CRISPR

Posted by Mary Gearing on Aug 10, 2017 10:19:54 AM

Any hint of CRISPR editing in human embryos has been met with a storm of media coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents another step towards CRISPR germline editing of disease-causing mutations. But how close are we really, and what new questions does this paper bring up? We’ll sift through the paper to understand what Shoukhrat Mitalipov and his colleagues have achieved, and how the field will move forward from this work.

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Topics: CRISPR

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