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Chlamydomonas single-celled algaIn this blog we will first overview the model system Chlamydomonas reinhardtii and methods for transforming this single celled, flagellated alga. We will provide protocol recommendations for high-efficiency square wave electroporation with generators such as BTX ECM 830, which can achieve a transformation efficiency of 2 to 6 x 103 transformants per µg of exogenous DNA. Next, we will cover protocol recommendations for exponential decay wave generators, such as the BTX ECM 630. Finally, we will compare the pros and cons and typical efficiencies of these methods to traditional glass bead transformation and electroporation via decaying square wave generators.  

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Posted By Michelle M. Ng-Almada, Ph. D.


T cells attacking a Cancer cellThis blog describes a new method for creating vaccines against tumors by Shadi-Yunger et al., 2019. Int. J. Cancer 144, 909-921. They report a novel MHC-II platform with a chimeric invariant chain, where the semi-peptide CLIP is replaced with a tumor specific peptide.  This hybrid MHC-II chain facilitates the activation of CD4+ T-cells when expressed in dendritic cells (DCs).  The researchers attempted to create an anti-melanoma vaccine by inserting different melanoma-associated antigens (MAAs) mRNA sequences into the MHC-II hybrid construct. Different MAAs were inserted into hybrid MHC-I and MHC-II constructs, and mRNAs were transfected into murine bone marrow derived dendritic cells (BMDCs) by electroporation using the BTX ECM 830 generator. Transfected BMDCs were injected into mice bearing melanoma tumors to assess the anti-tumor activity and T-cell activation stimulated by these modified DCs.  

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Posted By George Kamphaus, Ph. D.


Nerve cellsRegeneration of nerve cell axons is required for recovery of function after injury to the nervous system.  In this post we will review a method to directly trace regenerating axons in vivo, recently published by Gao et al. 2020. Neural Regeneration Research 23, 1160-1165. The researchers performed in vivo electroporation of mouse adult sensory neurons in the ipsilateral dorsal root ganglion to transfect plasmid DNA encoding enhanced green fluorescent protein.  Next, the sciatic nerve was squeezed with tweezers to create a model of sciatic nerve compression, and finally a direct time course of regenerating axon lengths was captured by confocal microscopy.  

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Posted By Michelle M. Ng, Ph. D.

 

T-Cell and Dendritic Cell Interactions

This blog describes a novel system for the study of T-cell activation by antigen-presenting dendritic cells (DC) and the use of a BTX ECM 830 electroporator to transfect quiescent (naïve or memory) T-cells. To increase the understanding of the intercellular dynamics of quiescent CD8+ T-cell interaction with DC and CD-4+ helper cells, Abu Shah, et. al. have created a 3-D system that reconstitutes a tissue-like environment that allows for the integration of cytokines, DC and quiescent T-cells. Utilizing electroporation to transfect mRNA constructs of modified antigen-specific TCRs, the researchers were able to express functional TCR on the surface of the quiescent T-cells and show activation by interaction with DCs loaded with the corresponding peptide antigen. 
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Posted By George Kamphaus, Ph. D.


Utilizing electroporation to brew a better beer

  • In honor of National Beer Day, April 7th, the focus of this blog post is how to use electroporation for genetic engineering of brewing yeast. First, we will provide the ideal electroporation parameters for Saccharomyces cerevisiae using either ECM 630 or Gemini X2 electroporators.  Then, we will highlight an example from the primary literature where this electroporation method was used with brewing yeast Saccharomyces cerevisiae ssp. carlsbergensis to modify the amount of ethanol in beer. 
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Posted By Michelle M. Ng, Ph. D.


Electrofusion Basics

There are different ways of analyzing efficiency following an electrofusion experiment. These methods vary in terms of how soon after electrofusion you may analyze results, what types of equipment and reagents are required, and what type of information is determined. In this post we will provide an overview of a couple of analysis methods that provide some initial results just minutes after electrofusion. 

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Posted By Michelle M. Ng, Ph. D.


CAR T-cell therapy

In this post we will highlight a high efficiency method for primary human T cell transfection utilizing electroporation of mRNA.  This method creates genetically engineered T-cells which specifically recognize antigens of interest for CAR T-Cell immunotherapy applications. 

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Posted By Michelle M. Ng, Ph. D.


Electrofusion Basics

The joining of membranes of neighboring cells by the application of a pulsed electrical field is called Electrofusion. Electrofusion is widely used for hybridoma creation for monoclonal antibody production, transgenics/somatic cell nuclear transfer applications, hybrid plant creation and functional studies combining two different cell types with different properties.  In this post we’ll explore the types of electrical wave forms used for electrofusion and outline the way that these waveforms are combined to make a successful electrofusion protocol. 

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Posted By Michelle M. Ng, Ph. D.


human brainIn this post we will review what organoids are and how they are used to create in vitro models to study organ function and human disease states in three dimensions. We'll cover the topic of organoid transfection via electroporation in more detail. Finally, we will highlight and summarize findings reported by Ogawa et al. 2018. Cell Reports 23, 1220-1229. These researchers utilized electroporation of CRISPR/Cas9 constructs to achieve gene modification of human cerebral organoids, resulting in a glioblastoma tumor model that could be further studied in vivo in xenografted mice.  

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Posted By Michelle M. Ng, Ph. D.


Electroporation Basics

In this post we will explore the relationship between electrical resistance and electrical conductance. We will also provide information on how resistance applies to electroporation applications, and finally we will answer a FAQ on the topic of sample resistance values in electroporation instrument log data. 

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Posted By Michelle M. Ng, Ph. D.
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