CRISPR-U™ Gene Knockout Cell Line Strategy

ACE2 Gene Knockout 266-6 Cell Strategy

CRISPR-U™ technology (CRISPR based), developed by Ubigene, is more efficient than general CRISPR/Cas9 technology in double-strand breaking and homologous recombination. With CRISPR-U™, Ubigene has successfully edited over 3000 genes on more than 100 types of cell lines.
Objective
To create a Human ACE2 Knockout model in 266-6 cell line by CRISPR-U™-mediated genome engineering.
Target gene info
Official symbol ACE2
Gene id 59272
Organism Homo sapiens
Official full symbol angiotensin I converting enzyme 2
Gene type protein-coding
Also known as ACEH
Summary The protein encoded by this gene belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases and has considerable homology to human angiotensin 1 converting enzyme. This secreted protein catalyzes the cleavage of angiotensin I into angiotensin 1-9, and angiotensin II into the vasodilator angiotensin 1-7. ACE2 is known to be expressed in various human organs, and its organ- and cell-specific expression suggests that it may play a role in the regulation of cardiovascular and renal function, as well as fertility. In addition, the encoded protein is a functional receptor for the spike glycoprotein of the human coronavirus HCoV-NL63 and the human severe acute respiratory syndrome coronaviruses, SARS-CoV and SARS-CoV-2, the causative agent of coronavirus disease-2019 (COVID-19).
Genomic regions Chromosome X
Cell line info
Cell name 266-6
Tissue pancreas
Organism Mouse
Morphology epithelial
Culture properties adherent
Disease pancreatic acinar cell tumor
Derivation 266-6 is an acinar pancreatic cell line derived in 1985 by Robert E. Hammer from a young adult mouse. The tumor was induced with an elastase I/SV-40 T antigen fusion gene.
Complete growth medium The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10% This medium is formulated for use with a 5% CO2 in air atmosphere. Standard DMEM formulations contain 3.7 g/L sodium bicarbonate for which a 10% CO2 in air atmosphere is recommended. ATCC tested fetal bovine serum is available as ATCC Catalog No. 30-2020 (500 mL) and ATCC Catalog No. 30-2021 (100 mL).
Subcultivation ratio Subcultivation Ratio:Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Cells must be seeded on 0.1% gelatin-coated culture vessels. To make the coating, dissolve 1.0 g of Gelatin Type A (Sigma G2500) in 1L of distilled water, sterilize by autoclaving. Add 0.4 to 0.5 mL of gelatin solution per cm2 of the culture vessel and refrigerate for 10 to 15 minutes. Prior to use remove the gelatin solution and rinse gently with culture medium, adding at least 0.1 to 0.2 ml per cm2. Discard the culture medium used for rinsing and use vessels immediately. If required, coated vessels can be prepared in advance and stored at 2 to 8˚C no more than 5 days. Remove and discard culture medium. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mMEDTA solution to remove all traces of serum that contains trypsin inhibitor. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes. Discard supernatant and resuspend cells in fresh culture medium.Add appropriate aliquots of cell suspension to new 0.1% gelatin coated culture vessels (see Handling Procedure for Frozen Cells). Incubate cultures at 37°C. Subculture Ratio: 1:3 to 1:4 Medium Renewal: 2 to 3 times a week. Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
Strategy Summary
This gene has 8 protein coding transcripts：
Name Transcript ID bp Protein Biotype CCDS UniProt Match RefSeq Match Flags
ACE2-202 ENST00000427411.2 7587 805aa Protein coding CCDS14169 Q9BYF1-1 - TSL:1, GENCODE basic, APPRIS P2,
ACE2-207 ENST00000678073.1 7487 805aa Protein coding CCDS14169 - - GENCODE basic, APPRIS P2,
ACE2-211 ENST00000680121.1 7413 805aa Protein coding CCDS14169 - - GENCODE basic, APPRIS P2,
ACE2-201 ENST00000252519.8 3339 805aa Protein coding CCDS14169 Q9BYF1-1 NM_001371415.1 TSL:1, GENCODE basic, APPRIS P2, MANE Select v0.92,
ACE2-205 ENST00000677282.1 6372 459aa Protein coding - - - GENCODE basic,
ACE2-210 ENST00000679278.1 3149 786aa Protein coding - - - GENCODE basic, APPRIS ALT2,
ACE2-206 ENST00000678046.1 2723 771aa Protein coding - - - GENCODE basic, APPRIS ALT2,
ACE2-209 ENST00000679212.1 2642 772aa Protein coding - - - GENCODE basic, APPRIS ALT2,
ACE2-208 ENST00000679162.1 2730 786aa Nonsense mediated decay - - - -
ACE2-203 ENST00000471548.5 998 112aa Nonsense mediated decay - - - CDS 5' incomplete, TSL:2,
ACE2-204 ENST00000473851.1 786 No protein Retained intron - - - TSL:3,
Ubigene Red Cotton Transcript
Strategy Click to get
Red Cotton™ Assessment    
Project Difficulty Level unknown
Cell Line 266-6
Target Gene ACE2
Colony Formation Intermediate
This KO Strategy loading
Red Cotton™ Notes Ubigene has successfully modified 266-6 cell line.
Aforementioned information comes from Ubigene database. Different origin of cell lines may have different condition. Ubigene reserved all the right for final explanation.
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