CRISPR-U™ Gene Knockout Cell Line
Strategy
ACE2 Gene Knockout MC3T3-E1 Subclone 14 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 MC3T3-E1 Subclone 14 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 | MC3T3-E1 Subclone 14 |
| Tissue | bone, calvaria |
| Organism | Mouse |
| Morphology | fibroblast |
| Culture properties | adherent |
| Derivation | A series of subclones were isolated from the cloned but phenotypically heterogeneous MC3T3-E1 cell line. |
| Complete growth medium | The base medium for this cell line is Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine and 1 mM sodium pyruvate, but without ascorbic acid (GIBCO, Custom Product, Catalog No. A1049001). 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. 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. Remove and discard culture medium. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53% (w/v) EDTA 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. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C Subculture Ratio: 1:6 to 1:8 Medium Renewal: Every 2 to 3 days. 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, |
Strategy
Click to get
Red Cotton™ Assessment
Project Difficulty Level unknown
| Cell Line | MC3T3-E1 Subclone 14 |
| Target Gene | ACE2 |
| This KO Strategy | loading |
| Red Cotton™ Notes | Ubigene has successfully modified MC3T3-E1 Subclone 14 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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Work flow
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