Name: removal of an internal translational start site from mrna while retaining expression of the full-length protein
Uploaded: 2022-03-16T13:30:00
Description: Watch this Scientific Journal Video about Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein at JoVE.com

The project was supported by National Institutes of Health grants (R01HL152691, R01HL138577, and R01HL159983) to RMS.

`All animal care and study protocols were approved by the Institutional Animal Care and Use Committees of Cedars-Sinai Medical Center and the University of Utah. C57BL/6J female mice obtained from commercial sources at 8-9 weeks of age (see Table of Materials) were used for the experiments.
1. Preparation for gene targeting
<ol>
	<li>Select the guide target sequence around the targeted mutation site coding M213 using CRISPOR web algorithm4<...

<ol>
	<li>Smyth, J. W., Shaw, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+of+connexin43+gap+junction+formation+by+internally+translated+isoforms.">Autoregulation of connexin43 gap junction formation by internally translated isoforms.</a> Cell Reports. 5 (3), 611-618 (2013).</li><li>Basheer, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GJA1-20k+arranges+actin+to+guide+Cx43+delivery+to+cardiac+intercalated+discs.">GJA1-20k arranges actin to guide Cx43 delivery to cardiac intercalated discs.</a> Circulation Research. 121 (9), 1069-1080 (2017).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cx43+isoform+GJA1-20k+promotes+microtubule+dependent+mitochondrial+transport.">Cx43 isoform GJA1-20k promotes microtubule dependent mitochondrial transport.</a> Frontiers in Physiology. 8, 905 (2017).</li><li>Xiao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxiliary+trafficking+subunit+GJA1-20k+protects+connexin-43+from+degradation+and+limits+ventricular+arrhythmias.">Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.</a> Journal of Clinical Investigation. 130 (9), 4858-4870 (2020).</li><li>Syvanen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gels+to+chips:+&amp;#34;minisequencing&amp;#34;+primer+extension+for+analysis+of+point+mutations+and+single+nucleotide+polymorphisms.">From gels to chips: &amp;#34;minisequencing&amp;#34; primer extension for analysis of point mutations and single nucleotide polymorphisms.</a> Human Mutation. 13 (1), 1-10 (1999).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+SNP+discovery+in+tetraploid+alfalfa+using+454+sequencing+and+high+resolution+melting+analysis.">Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis.</a> BMC Genomics. 12, 1-11 (2011).</li><li>Han, Y., Khu, D. M., Monteros, M. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+quick+PCR-based+method+to+genotype+mice+with+a+leptin+receptor+mutation+(db/db+mice).">A novel and quick PCR-based method to genotype mice with a leptin receptor mutation (db/db mice).</a> Acta Pharmacologica Sinica. 39 (1), 117-123 (2018).</li><li>Haeussler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR.">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biology. 17 (1), 148 (2016).</li><li>Concordet, J. P., Haeussler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPOR:+Intuitive+guide+selection+for+CRISPR/Cas9+genome+editing+experiments+and+screens.">CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.</a> Nucleic Acids Research. 46 (1), 242-245 (2018).</li><li>Hsu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Paquet, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+introduction+of+specific+homozygous+and+heterozygous+mutations+using+CRISPR/Cas9.">Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.</a> Nature. 533 (7601), 125-129 (2016).</li><li>Behringer, R., Gertsenstein, M., Nagy, K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2014).</li><li>Pomp, D., Critser, E. S., Rutledge, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+sodium+lactate+in+Whitten's+medium+improves+in+vitro+developmental+capacity+of+one-cell+mouse+embryos.">Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos.</a> Theriogenology. 29 (5), 1019-1025 (1988).</li><li>Summers, M. C., McGinnis, L. K., Lawitts, J. A., Raffin, M., Biggers, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IVF+of+mouse+ova+in+a+simplex+optimized+medium+supplemented+with+amino+acids.">IVF of mouse ova in a simplex optimized medium supplemented with amino acids.</a> Human Reproduction. 15 (8), 1791-1801 (2000).</li><li>Green, M. R. 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(95), e51879 (2015).</li><li>Iyer, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+unexpected+CRISPR-Cas9+off-target+activity+revealed+by+trio+sequencing+of+gene-edited+mice.">No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.</a> PLoS Genetics. 14 (7), 1007503 (2018).</li><li>Nature Medicine. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keep+off-target+effects+in+focus.">Keep off-target effects in focus.</a> Nature Medicine. 24 (8), 1081 (2018).</li><li>Mayer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+EcoRI-activity+of+EcoRI+endonuclease.">Optimization of the EcoRI-activity of EcoRI endonuclease.</a> FEBS Letters. 90 (2), 341-344 (1978).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutations+close+to+the+AUG+initiator+codon+affect+the+efficiency+of+translation+of+rat+preproinsulin+in+vivo.">Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo.</a> Nature. 308 (5956), 241-246 (1984).</li><li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point+mutations+define+a+sequence+flanking+the+AUG+initiator+codon+that+modulates+translation+by+eukaryotic+ribosomes.">Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.</a> Cell. 44 (2), 283-292 (1986).</li><li>Basheer, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+response+protein+GJA1-20k+promotes+mitochondrial+biogenesis,+metabolic+quiescence,+and+cardioprotection+against+ischemia/reperfusion+injury.">Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury.</a> JCI Insight. 3 (20), 121900 (2018).</li><li>Shimura, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+mitochondrial+fission+induced+by+stress-responsive+protein+GJA1-20k.">Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.</a> Elife. 10, 69207 (2021).</li></ol>

A gene-modified mouse model is a common approach for understanding gene function. However, since the GJA1-20k internally translated isoform is translated from the same Gja1 mRNA as full-length Cx43, a creative strategy was devised to retain full-length Cx43 expression yet suppress GJA1-20k expression. The approach is based on a mutation of the internal start codon of GJA1-20k. With a single point mutation, M213 was switched to L on Gja1 mRNA, which succeeded in suppressing GJA1-20k expression but retain...

The full-length Connexin 43 (Cx43) and the N-terminus truncated isoform, GJA1-20k, encoded by the same GJA1 mRNA but utilize different start codons1 to initiate translation. Cx43 translation occurs at the first AUG start codon, whereas GJA1-20k translation initiates at the AUG at residue 213. It was previously found that GJA1-20k has essential roles for full-length Cx43 trafficking, actin stabilization, and regulation of mitochondrial morphology in vitro1,2,3.
To understand the role of GJA1-20k in vivo, a GJA1-20k &#34;knock-out&#34; mouse model was generated that retained the ability to create full-length Cx43. The approach was to use the CRISPR-Cas9 system to substitute the single residue at 213 from a Methionine (M) to a Leucine (L) (Gja1M213L/M213L)4. An internal M to L mutation dramatically decreases the likelihood of internal translation occurring yet retains the translation and function of full-length protein4. Because the wild type (WT) and mutated allele have identical sizes and near-identical mRNA products, there is considerable difficulty in confirming genotype in the mice. DNA sequencing can identify the mutation but is too expensive and time-consuming for routine use. In general, several faster-genotyping methods have been established, such as Real-time Polymerase Chain Reaction (RT-PCR), minisequencing-ligation, high-resolution melting analysis, and tetra-primer amplification refractory mutation system PCR (ARMS-PCR)5,6,7,8. Yet these alternative methods require multiple steps, unique resources, and/or several specific primer sets which can induce non-specific PCR products.
This protocol introduces a detailed gene targeting and editing approach by CRISPR-Cas9 to create a single amino acid mutation, and rapid genotyping is presented to confirm the mutation. Genotype identification involves the creative use of restriction enzymes utilizing only a single set of primers to identify the target gene. Readers are referred to Reference4 to observe the profound electrophysiological effect of sudden cardiac death caused by a one residue Gja1M213L/M213L substitution mutation which still generates full-length protein yet fails to generate a smaller internally translated truncation isoform. This protocol will help make other mice models use a point mutation to decrease the internal translation of an isoform of interest while retaining the expression of the endogenous full-length protein.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL 8-Strip PCR tube</td>
			<td>Thomas Scientific</td>
			<td>1148A28</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>10x CutSmart buffer</td>
			<td>New England Biolabs</td>
			<td>B7204S</td>
			<td>Digestion buffer for NlaIII</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>50x TAE Buffer</td>
			<td>Invitrogen</td>
			<td>24710-030</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>Model # 9902</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>664</td>
			<td>mouse strain</td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>Bio-rad</td>
			<td></td>
			<td>To take gel image by 302 nm UV light</td>
		</tr>
		<tr>
			<td>eSpCas9 protein</td>
			<td>MilliporeSigma</td>
			<td>ESPCAS9PRO-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Lading Dye Purple (6x)</td>
			<td>New England Biolabs</td>
			<td>B7024S</td>
			<td>Loading buffer for electrophoresis</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hCG (human chorionic gonadotropin)</td>
			<td>MilliporeSigma</td>
			<td>23-073-425G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>MilliporeSigma</td>
			<td>H3884-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Lab software</td>
			<td>Bio-rad</td>
			<td></td>
			<td>To analyze gel image</td>
		</tr>
		<tr>
			<td>Inverted stereo microscope whit plasDIC</td>
			<td>Zeiss</td>
			<td>Axiovert A1</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA Mouse Genotyping Kits</td>
			<td>Roche Diagnostics</td>
			<td>KK7352</td>
			<td>For tissue or cell lysis, DNA extraction, and PCR master mix</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>LifeGlobal</td>
			<td>ZEKS-050</td>
			<td>embryo culture medium</td>
		</tr>
		<tr>
			<td>Lab coart</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>MilliporeSigma</td>
			<td>MR015D</td>
			<td></td>
		</tr>
		<tr>
			<td>NlaIII</td>
			<td>New England Biolabs</td>
			<td>R0125S</td>
			<td>To digest PCR product</td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td>To dilute reagents</td>
		</tr>
		<tr>
			<td>Paraffin oil</td>
			<td>Nacalai USA</td>
			<td>2613785</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugged 20 &#956;L fine pipette tip</td>
			<td>FisherScientific</td>
			<td>02-707-171</td>
			<td>To pick up blastocytes</td>
		</tr>
		<tr>
			<td>PMSG (pregnant mare&#8217;s serum gonadotropin)</td>
			<td>ProSpec-Tany</td>
			<td>HOR-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td>For pronuclear micorinjection buffer</td>
		</tr>
		<tr>
			<td>SYBR safe DNA Gel Stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td>To detect bands in gel</td>
		</tr>
		<tr>
			<td>tracrRNA</td>
			<td>Dharmacon</td>
			<td>U-002000-120</td>
			<td>Guid RNA</td>
		</tr>
	</tbody>
</table>

removal of an internal translational start site from mrna while retaining expression of the full-length protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

The CRISPR/Cas9 gene-editing system produces an ATG to TTA mutation and a silent TCC to TCG mutation at 56,264,279 to 56,264,281 and at 56,264,291 to 56,264,293 on mouse chromosome 10, or at 869 to 871 and 881 to 883 on Gja1 mRNA, respectively. Those mutation results in a Methionine 213 to Leucine (M213L) mutation on GJA1 protein and the TTC to TTG mutation disrupts a nearby PAM sequence to avoid undesired gene edition (Figure 1). The details of the mutation are described in a previous repor...

Watch this Scientific Journal Video about Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein at JoVE.com

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The present protocol describes a single M213L mutation in Gja1 that retains full-length Connexin43 generation but prevents translation of the smaller GJA1-20k internally translated isoform.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

The difficulty with a single residue substitution is to genotyping animals efficiently despite the difference in single residue. Special use of a restriction enzyme adds only a single, rapid, and low-cost step to typical PCR-based genotyping. This protocol can be applied to any other mouse model with a point of mutation if the target sequence is recognized by a restriction enzyme, which is usually the case.Cut one to three millimeters of the tail tip with clean scissors and transfer it to a 0.2-milliliter eight-strip PCR tube. After containing the tail sample as described in the manuscript, store it at T-minus 20 degrees Celsius until the extraction, up to about a week. Add 100 microliters of tissue lysis solution per tube and mix well, followed by spin-down using a tabletop minicentrifuge at 2, 200 times G for 10 seconds at room temperature.Ensure the tail samples are submerged in the solution. Set the tubes onto a thermal cycler set by programming the parameters for tissue lysis, inactivation, and holding. Store the tissue lysate at four degrees Celsius for up to a week if PCR cannot be performed immediately.Prepare a PCR solution containing five microliters of nuclease-free water, 0.75 microliters of 10-micromolar forward and reverse primers, and 7.5 microliters of PCR master mix per sample into a new PCR tube. Add one microliter of the previously prepared tissue lysate to the PCR solution. Mix the PCR solution well and spin down with a tabletop minicentrifuge at 2, 200 times G for 10 seconds at room temperature.Set the tubes onto a programmed thermal cycler. Once the reaction is completed, store the PCR products at four degrees Celsius for one to two months or approximately one year at minus 20 degrees Celsius. Prepare the enzyme solution containing seven microliters of nuclease-free water, two microliters of CutSmart buffer at 10X strength, and one microliter of NlaIII restriction enzyme.If there are several samples, multiply each content to make the mixture and aliquot 10 microliters per tube. Add 10 microliters of PCR product previously obtained to the enzyme solution per tube. Mix well and spin down with the tabletop minicentrifuge as demonstrated previously.Set the tubes onto a programmed thermal cycler or heat block. Store the product after incubation under cold conditions. Add 0.75 grams of agarose in 50 milliliters of single-strength TAE buffer.Mix and heat the agarose in the microwave until it dissolves completely. After cooling it down, add five microliters of DNA stain and gently mix. Pour 25 milliliters of the agarose gel into the gel mold and allow the gel to solidify.Load 10 microliters of digested PCR product to the well. Run the gel with 100 volts for 35 minutes. Image the agarose gel under ultraviolet light.The agarose electrophoresis performed using the digested PCR product resulted in several bands of different sizes in each genotype, with two bands in wild type, three in heterozygous, and one in homozygous, respectively. Test injection with blastocyst analysis resulted in a 76.9%success rate on injecting 50 nanograms per microliter of the donor oligos, and a 25%success rate on injecting 25 nanograms per microliter of the same donor oligos, respectively. Finally, injecting 50 nanograms per microliter of the donor oligos with ethanol precipitation obtained founder animals with a 50%success rate.Purification of oligo donors by ethanol precipitation lowered the toxicity while retaining high genome editing efficiency. Careful handling and in adding a low amount of reagents is very important. Ensure they are correctly added and are mixed well.

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Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...