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Crispr cas9 technique

Manufactured by Cyagen
Sourced in China

The CRISPR/Cas9 technique is a gene-editing tool that allows for precise modification of DNA sequences. It utilizes a guide RNA and the Cas9 enzyme to target and edit specific genetic regions.

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5 protocols using crispr cas9 technique

1

Generation and Characterization of Trim40 Knockout Mice

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C57BL/6N wild-type male mice were obtained from the OrientBio and Trim40-/- male mice were generated on C57BL/6N background using CRISPR/Cas9 technique from Cyagen Biosciences. CRISPR guide RNA (gRNA) was used to target exon 1 region in the Trim40. Genotypes of mice were investigated using genomic DNA PCR. All mice were maintained in the specific pathogen-free facility of the Yonsei Laboratory Animal Research Center at Yonsei University according to Korean Food and Drug Administration. All mice were housed on a 14-hour light and 10-hour dark cycle with 18–23 °C and 40–60% humidity and were maintained in ventilated cages under standard housing conditions with access to food and water ad libitum. No more than 5 mice were housed in cages and all mice used in this study were appropriately monitored daily changes in body weight, food or water intake, and physical appearance. At experimental endpoints, mice were euthanized with CO2 inhalation for at least 5 min until breathing and heartbeat stopped. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei University (YLARC, No. IACUC-202011-1177-01, IACUC-202107-1302-02, IACUC-202107-1302-03, IACUC-202204-1447-01, IACUC-202204-1448-01, IACUC-202209-1541-01 and IACUC-A-202209-1542-01).
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2

BALB/c and TLR4 KO Mice DNA Vaccination

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Six to eight weeks old female BALB/c mice (Guangdong Medical Laboratory Animal Center) and TLR4 knockout C57BL/6 mice generated using CRISPR/Cas9 technique (Cyagen Biosciences) were bred under standard pathogen-free conditions. For DNA vaccination, each mouse was immunized with 20 μg of endotoxin-free DNA vaccines or pVAX1 vector twice at four-week intervals intramuscularly plus electroporation as we described previously [13 (link)]. Two weeks after the last immunization, mice were euthanized for immunogenicity analysis. For tumor challenge, two weeks after the 2nd immunization, 5 × 105 AB1-Gag cells were inoculated subcutaneously in the right flank of mice. The tumor size was measured each two-four days. Tumor size based on caliper measurement was calculated by the modified ellipsoidal formula, tumor size = (length × width2)/2. Mice were sacrificed at the endpoint (21 days post-challenge). The mice were raised under specific pathogen-free (SPF) conditions and were fed a normal diet.
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3

Establishing and Characterizing LBH-Knockout Mouse Model for Myocardial Infarction

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All animal procedures were designed and performed according to the regulations of the Institutional Animal Care and Use Ethics Committee of Zhujiang Hospital. The establishment of conventional LBH- KO C57BL/6 mice was performed by Cyagen, Inc., via the CRISPR/Cas9 technique (contract ID: KOAI200813MG1) to generate F0 founder animals; then, the wild-type (WT)/KO offspring were bred, identified, and maintained at the Animal Experiment Center of Zhujiang Hospital (Figure S1). The MI model was constructed according to our previous research [13 (link)]. All mice were sacrificed to obtain the hearts on days 2 and 3 after surgery. Each experimental group had at least four surviving mice for sampling at every time point. Paraffin-embedded tissue sections of the left ventricles were used for hematoxylin and eosin (H&E), Masson, and immunofluorescence staining (Figure S2), and the remaining peri-infarct tissues were used for western blot (WB) assays.
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4

Generation and Validation of Tmem150b Knockout Mice

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Tmem150b knockout (KO) mice were constructed by CRISPR/Cas9 technique (Cyagen, Suzhou, China) with deletion of 989 bp fragment encompassing exons 2–4. Genotyping was performed by PCR from genomic DNA of mouse tails. Validation of gene knockout in mRNA level was done by RT-PCR using cDNA obtained by reverse transcription of mRNA from mouse ovaries and the PCR products were confirmed by Sanger sequencing. The sequences of primers were as follows: wild type (WT) allele, F: 5′-GACTGCTTGGAGATCCAGCT-3′, R: 5′-GTGGAGGCAGTCTGACTATC-3′; and delete allele, dF: 5′-CTTTGTGCCCTGGGTACCTC-3′, R: 5′-GTGGAGGCAGTCTGACTATC-3′; F’: 5′- AGTGGGACCACAAAAGCAGG-3′, R’: 5′- CCTTTCACCCACCAGGACAG-3′. All PCR products were separated by 1.5% (w/v) agarose gel, stained with ethidium bromide (Invitrogen, USA) and detected by Chemidoc MP System (Bio-Rad, USA). All genetically altered mice had a mixed background of ICR and C57BL/6J. WT mice for experiments were purchased from Charles Rive Company. Mice were housed in a temperature-controlled (22 ± 2 °C) room with a 12/12 h light/dark cycle and free access to water and food. All animal experiments were performed in accordance to the ethical guidelines approved by Animal Care and Research Committee of Shandong University.
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5

Generation of Knock-in Mouse Models

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Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ knock-in mice were constructed by the CRISPR/Cas9 technique (Cyagen, Suzhou, China). The gRNAs, donor oligos containing each mutant, to Plxnb2 (p.D1577E and p.R465Q) or Ryr2 (p.N1552S and p.R137W) gene, and Cas9 was coinjected into fertilized mouse eggs to generate targeted knock-in offspring. Genotyping was performed by PCR from genomic DNA of mouse tails followed by Sanger sequencing. The method is shown in Supplementary Fig. S1, and the primers are shown in Supplementary Table SII. All genetically altered mice had a background of C57BL/6J. WT mice for experiments were purchased from the Beijing Vital River Laboratory Animal Technology Company. Mice were housed at 22 ± 2°C controlled room temperature with a 12/12 h light/dark cycle and free access to water and food. All animal experiments were performed in accordance with the ethical guidelines approved by the Animal Care and Research Committee of Shandong University. From the F2 generation, Ryr2N1552S/+ and Ryr2R137W/+ mice were mated to each other and WT mice, and the offspring genotypes were validated by sequencing using corresponding primers. The Plxnb2D1577E/+ and Plxnb2R465Q/+ point mutant mice were mated in the same process. The detailed methods of reproductive performance and fertility testing are recorded in the Supplementary Materials and methods.
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