Creative Biolabs offers conventional knock-out models using either the most commonly used ESC/HR-based gene targeting strategies or the latest nuclease-mediated gene editing technologies, including CRISPR/Cas9 and TALENs.
What Is A Conventional Knock-out Mice?
Conventional knock-out mouse models are characterized by the targeted deletion of one or more genes in all tissues at all times. The targeted gene is replaced with a foreign DNA sequence, usually, a drug selection marker, which is responsible for drug resistance and thus is used to positively select cells that have integrated the targeting vector into their chromosome. Cells that are integrated with the selectable marker will survive the treatment of neomycin, which is the most commonly used aminoglycoside antibiotic for positive selection. The lab-made construct to replace the mouse endogenous gene is often called the targeting construct.
The Pros and Cons of Conventional Knock-out Models
The greatest strength of the conventional knock-out mouse model is that it bears complete inactivation of the gene in all expressing cells at every expressing time point in development, allowing for studies of the physiological or pathological functions of a gene at a systemic level. Also, the design of targeting strategy and targeting construct are simple.
However, many drawbacks are inherited. The first is this approach is only practical for knock-out genes that don't cause a lethal phenotype. When the gene is crucial for development, it may not be suitable to choose this method. What's more, sometimes phenotype can be abnormal even at the heterozygous stage and expression dysregulation of related genes can occur. When attempts are to be made at genes located on the X-chromosome, careful considerations are needed because males will only have one copy of the gene and may be severely affected by the knock-out. Besides, the molecular cloning of the targeting construct can be labor intensive.
A marker gene, typically neor, is integrated into the genome of isolated embryonic stem (ES) cells, followed by positive selection of the successfully incorporated cells with neomycin. These cells are inserted into a mouse blastocyst and are then implanted into the uterus of female mice, thereby producing chimeras. When these heterozygous offspring are interbred, some of their offspring will inherit the knock-out gene from both parents, called homozygous KO models.
CRISPR/Cas9-Based Targeting Strategy
Apart from ESC/HR based gene targeting strategy, conventional deletions can also be achieved using CRISPR/Cas9 technology, either by microinjection or via CRISPR-based technology in ES cells.
Our technical team is expert in the assessment of project feasibility and is willing to assist you in the right selection of the targeting strategy that would best meet your project goals.
Meanwhile, Creative Biolabs offers other types of knock-out models that you may be interested in:
Equipped with both cutting-edge technology platforms and specialists in this field, Creative Biolabs is capable of offering our clients the most reliable model creation services at most favorable prices currently on the market. Contact or inquire us to discuss the ideal custom mouse model to boost the impact of your next project and beyond.
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