Name: production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex
Uploaded: 2018-01-09T12:30:00
Description: Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Research reported in this publication was supported by the National Institutes of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health under award number 1R41OD021979 (to ZW) and by a research grant from the Next-Generation BioGreen 21 Program, Republic of Korea, grant no. PJ01107704 (to ZW) and grant no. PJ01107703 (to IK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or BioGreen 21. We thank Dr. Nikolas Robl for editing the manuscript.

The procedures described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of Utah State University (IACUC protocol: 2484). Hamsters used in this protocol are adult (6 - 10 weeks of age) LVG strain golden Syrian hamsters. All hamsters are housed in the vivarium at the Bioinnovation center, Utah State University. Room temperature is set at 23 &#176;C, humidity is set at 40 - 50%, and light cycle is set 14L:10D (light:dark). Whenever possible, embryo manipulation and surugical proced...

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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=Syrian+hamster+as+a+permissive+immunocompetent+animal+model+for+the+study+of+oncolytic+adenovirus+vectors.">Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors.</a> Cancer Res. 66 (3), 1270-1276 (2006).</li><li>Thomas, M. A., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Syrian+hamster+as+an+animal+model+for+oncolytic+adenovirus+vectors.">Use of the Syrian hamster as an animal model for oncolytic adenovirus vectors.</a> Methods Mol Med. , 169-183 (2007).</li><li>Hogarth, C. A., Roy, A., Ebert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+evidence+for+the+absence+of+a+functional+cholesteryl+ester+transfer+protein+gene+in+mice+and+rats.">Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats.</a> Comp Biochem Physiol B Biochem Mol Biol. 135 (2), 219-229 (2003).</li><li>Ebihara, H., 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+Syrian+golden+hamster+model+recapitulating+ebola+hemorrhagic+fever.">A Syrian golden hamster model recapitulating ebola hemorrhagic fever.</a> J Infect Dis. 207 (2), 306-318 (2013).</li><li>Jove, 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=Lipidomic+and+metabolomic+analyses+reveal+potential+plasma+biomarkers+of+early+atheromatous+plaque+formation+in+hamsters.">Lipidomic and metabolomic analyses reveal potential plasma biomarkers of early atheromatous plaque formation in hamsters.</a> Cardiovasc Res. 97 (4), 642-652 (2013).</li><li>Vairaktaris, E., 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=The+hamster+model+of+sequential+oral+oncogenesis.">The hamster model of sequential oral oncogenesis.</a> Oral Oncol. 44 (4), 315-324 (2008).</li><li>Paciello, O., 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=Syrian+hamster+infected+with+Leishmania+infantum:+a+new+experimental+model+for+inflammatory+myopathies.">Syrian hamster infected with Leishmania infantum: a new experimental model for inflammatory myopathies.</a> Muscle Nerve. 41 (3), 355-361 (2010).</li><li>Safronetz, D., Ebihara, H., Feldmann, H., Hooper, J. 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To better exploit the potential of golden Syrian hamsters as models of human disease, we developed a PN injection protocol for delivering a CRISPR/Cas9 complex to target the hamster genome. The PN injection protocol optimizes several key variables including the embryo culture medium, temperature, and wavelengths of light13. There are also several hamster-specific animal handling procedures that need to follow for successfully conducting gene targeting in the hamster. For example, sexually matured ...

The golden Syrian hamster (Mesocricetus auratus) is one of the most widely used rodents for biomedical research. According to the U.S. Department of Agriculture, approximately 100,000 hamsters were used in the United States in 2015, representing 13% of total laboratory animal usage among the species covered by the Animal Welfare Act (http://www.aphis.usda.gov; accessed March 10, 2017).
The hamster offers several advantages over other rodents in the study of a number of human diseases. For example, the histopathology of N-nitrosobis(2-oxopropyl)amine (BOP) induced pancreatic ductal adenocarcinomas in hamster is similar to human pancreatic tumors, while BOP treatment mainly induces thyroid gland tumors in rats and lung and liver tumors in mice1. Because hamsters are the only small rodent found to support the replication of adenoviruses, they are also the model of choice for testing adenovirus-based oncolytic vectors and anti-adenovirus drugs2,3,4. Another example wherein the hamster model offers an advantage over mice and rats is in the study of hyperlipidemia. Humans and hamsters exhibit great similarities in lipid metabolic pathways and both species carry the gene encoding cholesteryl ester transfer protein (CETP), which plays a central role in lipid metabolisms, while CETP is absent in mice and rats5. Additionally, hamsters develop hemorrhagic disease more representative of the human manifestation following exposure to Ebola virus6. Hamsters are also the models of choice for studying atherosclerosis7, oral carcinomas8, and inflammatory myopathies9. Recently, it has also been demonstrated that hamsters are highly susceptible to Andes virus infection and develop hantavirus pulmonary syndrome-like disease, providing the only rodent model of Andes virus infection10.
To address the unmet need for novel genetic animal models to study the human diseases where no reliable small rodent model is available, we recently have succeeded in applying the CRISPR/Cas9 system to the hamster and have produced several lines of genetically engineered hamsters11. Hamster zygotes are highly sensitive to environmental milieus such that the PN injection protocols developed in other species are unsuitable. Therefore, we developed a PN injection protocol for the hamster that accommodates the special requirements for handling hamster embryos in vitro. Here, we describe the detailed PN injection procedure using the CRISPR/Cas9 system and the accompanying steps, from the preparation of single guide RNA (sgRNA) to the transfer of injected embryos into recipient females.

<table>
	<tbody>
		<tr>
			<td>Cas9</td>
			<td>Invitrogen</td>
			<td>B25640</td>
			<td>1 ug/ul (~6.1 uM)</td>
		</tr>
		<tr>
			<td>GeneArtTM Precision Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Albumin from human serum</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td>For cultivation medium</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>NI-150</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>New Brunswick</td>
			<td>Galaxy 14S</td>
			<td>For embryo cultivation</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>PB-7</td>
			<td>For making injection needles</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti-S</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>invitrogen</td>
			<td>SMZ745T</td>
			<td>For embryo transfer</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M1840</td>
			<td>Keep in dark</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4877-2000IU</td>
			<td>For superovulation</td>
		</tr>
	</tbody>
</table>

production of genetically engineered golden syrian hamsters by pronuclear injection of the crispr/cas9 complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

The efficiency of the described protocol in producing genetically modified hamsters depends on the outcomes of the following two critical steps: the live birth rate of recipient females and the number of live pups with the intended genetic modifications. The live birth rate is a direct results of the embryo quality and the skill of the individual performing the PN injection and embryo transfer procedures. To ensure that the developmental potential of manipulated embryos is not compromised...

Watch this Scientific Journal Video about Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex at JoVE.com

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

The overall goal of this protocol is to provided viewers with detailed procedures to produce Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex. This PN injection protocol we developed in the Golden Syrian Hamster makes genetic engineering in this business possible. This technology is a great contribution to the field of animal model development.Due towards extreme sensitivity of hamster embryos to the environment, we developed a unique pronuclear injection protocol to accommodate the special niche of hamster embryos. The development of this technique is a great step forward in human disease modeling, because for the first time, genetically engineered hamster models can be produced. For this experiment, one day prior to pronuclear or PN injection, prepare Zygote handling dishes and PN injection drop in a lid of a 35 millimeter dish with HECM-9 medium.Cover the medium drops with mineral oil. To balance the drops and HECM-9 medium, incubate it at 37.5 degrees celsius overnight. On the day of PN injection, prepare embryo-flushing dishes using HECM-9.Store them in the incubator until needed. To isolate oviducts, place a euthanized superovulated female hamster in dorsal recumbency on a surgical drape. Scrub the abdomen with 70%ethanol.Next, firmly hold skin and abdominal muscle layer at the midline, and make an incision along the end of the abdomen. To expose the abdominal organs, incise the peritoneum. Next, use a fine forceps to grasp one of the uterine horns, and retract the tissue from the abdomen.Separate the uterus from the mesometrium and fat tissue. Make the first cut between the oviduct and ovary and a second next the oviduct-uterus junction while including a small part of the uterus. Place the oviducts in the same flesh dish.To collect zygotes under the dissection microscope, use a number five Dumont forceps to grasp the side of the uterine horn. Insert a homemade needle into the lumen of the oviduct from the infundibular end. Then, flush the oviduct with 300 to 400 microliters of HECM-9 medium.Immediately after flushing, collect the zygotes;and wash them twice with HECM-9 in the handling dish. All zygotes should be denuded from the cumulous cells at this stage of development. Right after that, place the zygotes in an incubator until the injection to avoid exposure to light.To prepare for injection, thaw the Cas9-sgRNA RNPs on ice. Immediately before the injection, load the injection needle with RNP solution by placing the rear end of the needle into the tube containing the solution allowing it to fill the needle by capillary action Next, fill the holder pipette with mineral oil up to approximately three millimeters of the needle bend. Place the holder in the micro injector with the tip of the holder pipette horizontal to the bottom of the dish.Fill the needle tip with medium by suction. Then, set the injection needle at a 10 to 15 degree angle opposite to the holder. Next, identify a zygote, and use the holder needle to apply a fine suction.Male, which is slightly bigger, and female pronuclei should preferably be within the same focal range and aligned parallel to the holder. Using the injection needle at a three o'clock position, penetrate the zona pellucida and male pronuclei. Once the pronucleus swells, withdraw the injection needle quickly to prevent the nucleus from adhering to the needle and to avoid damaging it.Efficiency of gene targeting for STAT2 and RAG1 genes by the Cas9/sgRNA system is evaluated by live birth rate of recipient females and the number of live pups with the intended genetic modifications. Genotyping results from a PCR restriction fragment length polymorphism assay, or PCR-RFLP, of pups, show the efficiency of gene targeting. Lane one indicated biallelically targeted alleles, and lanes three and seven show monoallelically, or mosaically, targeted alleles.This technology has paved the way for researchers to develop newer genetically engineered hamster models of human disease. After watching this video, you should have a good understanding of how to produce genetically engineered Golden Syria Hamsters by a pronuclear injection of the CRISPR/Cas9 Complex.

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Research

JoVE Journal

Bioengineering

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different techniques and varies in price, materials, and purpose. Many of these fabrication methods result in polydisperse populations with non-uniform activation thresholds. Additionally, controlling the droplet sizes typically requires stable perfluorocarbon liquids with high activation thresholds that are not practical in vivo. Producing uniform droplet sizes using low-boiling point gases would be beneficial for in vivo imaging and therapy experiments. This article describes a simple and economical method for the formation of size-filtered lipid-stabilized phase-shift nanodroplets with low-boiling point decafluorobutane (DFB). A common method of generating lipid microbubbles is described, in addition to a novel method of condensing them with high-pressure extrusion in a single step. This method is designed to save time, maximize efficiency, and generate larger volumes of microbubble and nanodroplet solutions for a wide variety of applications using common laboratory equipment found in many biological laboratories.

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...