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14 results for “Microinjection”
Protocol for transfection by microinjection into the eggs of the parasite vector snail Biomphalaria glabrata
<p><strong>1. Egg production</strong></p> <p>Place about 30 adult snails (10 mm diameter) into a 5.5-liter water tank. Place a piece of polystyrene of (3 x 3 cm) in each tank. There is the preferred support of <em>Biomphalaria glabrata</em> for laying its eggs. The snails are fed <em>ad libitum</em> with green lettuce leaves, they can also be fed with dry spirulina to boost reproduction. Maintain water at a temperature of 25 degrees Celsius.</p> <p><strong>2. Egg collection</strong></p> <p>Gently pick up several egg layers from the polystyrene with soft holding forceps and place the eggs into a petri dish with natural mineral water (e.g. Volvic) to prevent them from drying out.</p> <p>Start sorting the eggs under the stereoscopic microscope to choose only the gastrula stage and place them into another petri dish with natural mineral water.</p> <p> </p> <p><strong>3. Preparation of the transfection solution</strong></p> <p><strong>Material:</strong></p> <p>a. <em>in vivo</em> JetPEI transfection reagent</p> <p>b. 10% glucose solution</p> <p>c. 5% glucose solution</p> <p>d. Plasmids (dCas9-SunTag-BFP and scFv-DNMT3A-GFP)</p> <p>e. 0.2 ml microtubes</p> <p>f. P10 and P200 pipettes</p> <p>g. P10 and P200 pipette tips</p> <p>h. Permanent marker</p> <p>The glucose solution and the <em>in vivo</em> jetPEI transfection reagent are equilibrated at room temperature. </p> <p>Prepare 21 µl of each plasmid at a concentration of 78 and 88 ng / µl respectively (for a total volume of 42 µl =equals 3.5 µg of DNA) add the plasmid DNA to a 0.2 ml tube (labeled as Tube A) and mix with 21 µl of 10% glucose solution. </p> <p>In another microtube (labeled as Tube B), add 21 μl of 5% glucose solution and 1 μl of <em>in vivo</em> jetPEI. </p> <p> Prepare a third tube (labeled as Tube C) with 21 µl of 5% glucose solution and 0.5 µl of <em>in vivo</em> jetPEI to inject into embryos that will serve as controls. </p> <p>Leave the solutions at room temperature while you prepare the microinjection station. </p> <p> </p> <p> </p> <p><strong>4. Preparation of the micro-injection station</strong></p> <p><strong>Material:</strong></p> <p>a. Pre-pulled glass micropipettes (1mm diameter)</p> <p>b. Watch glass</p> <p>c. Modeling clay</p> <p>d. 35 mm and 90 mm petri dishes</p> <p>e. Mineral oil (M5904, SIGMA)</p> <p>f. Wash bottle with natural mineral water (Volvic)</p> <p>g. 0.2 ml microtubes</p> <p>h. 12-well cell culture plate</p> <p>i. Fine brush</p> <p>j. Phenol red solution</p> <p>k. Pasteur pipette or dropper</p> <p>l. Dissection forceps</p> <p>m. Soft holding forceps</p> <p>n. Snail eggs in the gastrula stage</p> <p>o. Drummond Scientific Nanoject III Programmable Nanoliter Injector</p> <p> </p> <p>Take a pre-pulled glass micropipette and cut it with a scalpel to have a ~ 0.2 mm tip slightly beveled if possible.</p> <p>Before attaching the micropipette to the programmable nanoliter injector, fill it with mineral oil. If this step is not done, the injector will not work properly. This can be done with a filling needle </p> <p>attached to a hamilton syringe of 10 microliters.</p> <p>When the micropipette is filled with oil, it must be fixed on the injector. For this it is necessary to: </p> <p>Slide the chuck and collet onto the glass micropipette, then slide the black O-ring with the seal onto the wire plunger </p> <p>With the micropipette attached to the injector, press the [EMPTY] icon until the plunger is fully extended. This step can be done with the footswitch by pressing once [EMPTY] then [STOP] and then proceeding [EMPTY] with the foot switch. A single beep is emitted when the plunger is fully extended.</p> <p>Fill the micropipette with 3 µl of the control solution or the transfection solution by placing the glass micropipette tip in a 0.2 ml tube with the solution to be injected and pressing the [FILL] icon. It is desirable to fill it at a slow rate, by pressing the [FILL] icon for a few seconds, then the [STOP] icon to allow the sample to equilibrate before pressing again the '[FILL] icon.</p> <p>Note: The piston continues to extend or retract until the [STOP] icon is pressed, or until the fully extended or fully retracted position is reached.</p> <p> </p> <p>5. Microinjection</p> <p>Place a watch glass into a 35mm petri dish and secure it on one side with modeling clay to form a slope. Use soft handling forceps to transfer an egg mass and lay it on the slope side of the watch glass so that the egg mass is in a sloping position.</p> <p>Remove excess water from the eggs with absorbent paper. Rehydrate if necessary with a fine brush to improve the visibility of the embryos. To inject the sample, return to the operating mode screen by pressing the [EXIT] icon, then select the injection mode by pressing the [INJECT] icon. Set the injection volume to 30nL and the flow rate to 20nL per second using the icons [+] and [-] respectively. Press the [INJECT] icon to inject the sample. </p> <p>Inject 30nL of the microinjection solution into each egg. Place the microinjected egg masses in a 12-well cell culture plate and note with a marker whether they were microinjected with the control solution or with the solution containing the plasmids.</p> <p>We colored the injection solution with red phenol to facilitate the visibility in this video.</p> <p><strong>Monitor the expression of the plasmids</strong></p> <p>Monitor the plasmids expression 72 h after microinjection in a contrast / fluorescent microscope or in a fluorescent stereo microscope. Then sort the fluorescent snails and perform a second micro-injection with a solution containing 10 µl of single guide RNA (at a concentration of 2ng / µl), add 0.5 µl of <em>in vivo</em> jetPEI reagent and 10 µl of 5% glucose solution. 3 days after the second microinjection, collect the hatched snails in a 1.5 ml tube containing 25 µl of lysis buffer for DNA and RNA purification.</p> <p>In this photo produced under a confocal microscope we washed a veliger larva in PBS solution, then we fixed it with 4% paraformaldehyde solution and then we placed it in a slide with two drops of the Dako fluorescence mounting medium. </p> <p>96 after the transfection we can observe the expression of the green fluorescent protein, the blue fluorescent protein and the co-localization of both proteins. </p> <p>This protocol is used to perform DNA methylation changes in a target gene. This transfection protocol can be used with other plasmids, with small interfering RNAs, or with messenger RNAs.</p> <p>Produced at IHPE (http://ihpe.univ-perp.fr)</p>
Short-term effects of human versus bovine sialylated milk oligosaccharide microinjection on zebrafish larvae survival, locomotor behavior and gene expression
GEO Series GSE218857. Danio rerio. 9 samples. Type: Expression profiling by high throughput sequencing.
Dual Host-Pathogen RNA-Seq Aphanius dispar and Candida auris Yolk Sac Microinjection
GEO Series GSE277854. blank sample; Candidozyma auris; Aphanius dispar. 57 samples. Type: Expression profiling by high throughput sequencing.
Microinjection of human mesenchymal stem cells to mouse blastocysts
GEO Series GSE195573. Mus musculus; Homo sapiens. 14 samples. Type: Expression profiling by high throughput sequencing.
Comparison of Intradermal Microinjections of Tanexamic Acid and Oral Tranexamic Acid in the Management of Melasma.
ClinicalTrials.gov study NCT07280234. IPD Sharing: UNDECIDED. Countries: 1. Publications: 0.
Restylane Silk Microinjections to Cheeks
ClinicalTrials.gov study NCT03742479. IPD Sharing: NO. Countries: 1. Publications: 0.
Evaluation of Microinjected Amnion-derived Collagen by Dermapen in the Smoothing of Skin Wrinkles
ClinicalTrials.gov study NCT06054646. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Direct Tumor Microinjection and FDG-PET in Testing Drug Sensitivity in Patients With Relapsed or Refractory Non-Hodgkin Lymphoma, Hodgkin Lymphoma, or Stage IV Breast Cancer
ClinicalTrials.gov study NCT03432741. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Transcriptome changes in bovine oocytes caused by microinjection
GEO Series GSE139867. Bos taurus. 24 samples. Type: Expression profiling by high throughput sequencing.
Microinjection of human ESC-derived trophoblast cells(BAP cells) to mouse blastocysts
GEO Series GSE224029. Homo sapiens. 6 samples. Type: Expression profiling by high throughput sequencing.
Comparing the Transcriptomes of Marf1-genetrap (GT) oocytes with those microinjected with mRNAs for wild type MARF1 (GTWT) or D272-mutated MARF1 (GTD272) by RNA-Seq Analysis
GEO Series GSE109195. Mus musculus. 12 samples. Type: Expression profiling by high throughput sequencing.
Zebrafish embryos microinjected with ptGFP1 (control group) or ptGFP1-NOD1 (NOD1 group)
GEO Series GSE116109. Danio rerio. 6 samples. Type: Expression profiling by RT-PCR.
Supplementary material for Comparison between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-outs
<p>Data upload for my dissertation about gene editing</p>
Supplementary Material for Comparison between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-outs
<p>Data upload for my dissertation about gene editing</p>
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