Our innovative gene-editing technology increases gene-editing efficiency by 10 to 20 fold, making our custom model development process faster and more affordable for your research.
Gene editing services assist researchers in studying the function of genes and to associate them with physiological and pathological phenotypes. Gene editing services are also often used to generate animal models of diseases which can lead to the development of novel therapies. CRISPR/Cas9 based gene editing is employed to create humanized animal models which are widely used for various diseases, drug screening, organ transplantation, diagnostics, and personalized treatments, most notably in the field of oncology.
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In the past decade, Biocytogen has grown into a global leader in the field of gene editing services and animal/cell model generation. Our cutting-edge technology platforms enabled our vast collection of humanized mouse models for research and drug screening/evaluation, and offers custom in vitro and in vivo models. We generate approximately 800 in vivo models every year and ship to our customers worldwide. Biocytogen provides outstanding technical support, customer service, products with premier quality, and quick turnaround times. For more information about our gene editing services and animal model generation, contact us or see a message from our experts about our strict Quality Control measures.
Based on specific project goals, our expert scientists will work with you to select the optimal technologies you can employ to produce disease models or advanced humanized models. With varied success rate requirements, experimental time lines, and resource allocations, please consult our team for a specialized experimental design best suited for your needs.
Biocytogen's professional technology team and sales staff strive to help researchers achieve their animal model needs. The gene targeting strategies that our customized models use include:
In a global knockout (KO) mouse model, an exon of a target gene is globally deleted (EGE™ method) or replaced (ESC/HR) with a positive selection marker (Neomycin in most cases), thus inactivating the gene. In a global, or whole-body KO mice, the gene of interest in disrupted in every tissue.
Global knockin mouse models introduce mutant or exogenous DNA sequences into a specific locus in the mouse genome. These models can mimic genetic diseases when mutation(s) are introduced, or monitor gene expression when genes are tagged with various proteins (e.g. EGFP, mRFP, mCherry, YFP, LacZ, and Flag).
A conditional knockout (cKO) model is generated via Cre-LoxP/Flp-Frt recombination systems. The targeted fragment to be knocked out is flanked by LoxP (or Frt) elements. Floxed mice are then bred with tissue-specific Cre or Flp mice, and sequences between the LoxP sites will be removed from the offspring’s genome in a tissue-specific pattern. LoxP fragments are typically inserted into the introns downstream of the ATG-containing exon, thereby removal of the flanked exon(s) will result in a frame-shift that disrupts protein expression.
In a conditional knockin (cKI) mouse model, mice containing a floxed (or conditional) allele are crossed with Cre-expressing mice, allowing for tissue-specific expression of any exogenous elements, including mutation, reporter, etc. Conditional knockin mice are usually generated using either FLEx (Flip Excision) approach (also known as dual-lox system) or minigene system. After Cre-lox recombination by having conditional knockin mice bred with tissue specific Cre or CreERT2 mice, the mutation/reporter will be expressed in a spatial and/or temporal manner.
A conventional point mutation mouse model is a knockin mouse line in which one or more nucleotides in the mouse genome are substituted by variant nucleotides. This can result in either an in-frame amino acid change within a given protein sequence, or a frameshift mutation. Knockin point mutation mouse models are widely used to study the roles of particular nucleotides or amino acids within proteins, which is directly applicable for studying human genetic diseases.
A conditional point mutation mouse model introduces a point mutation when certain conditions are met. In the gene targeting strategy below, when Cre recombinase is present, a point mutation will be introduced tissue specifically via Cre activity. There are two major design strategies illustrated below.
Traditional transgenic mouse models are generated via pronuclear injection of a plasmid, where many different founders can be obtained. Experimental results from different founders can vary and not be reproducible due to the differences in integration copy number and loci. Currently, most researchers use site-specific integration strategies to build gene edited mouse models. Rosa26 is the most commonly used “safe harbor” locus because Rosa26 encodes a nonessential nuclear RNA expressed in almost all tissues. Conditional expression of an exogenous gene will result when a LoxP-3XSTOP-LoxP sequence is inserted upstream of the exogenous sequence at the Rosa26 locus, and this model is crossed with a Cre deleter. Examples of additional safe harbor loci include H11 and TIGRE.
Genes tagged with EGFP, YFP, LacZ, Flag, mCherry and other sequences are useful for monitoring gene expression. Reporter gene mouse models are used to construct phylogenetic trees for cell development studies. Replacement of an endogenous gene with a reporter can simultaneously achieve gene knockout and knockin in the same mouse model.
The Tol2 mouse model allows generation of transgenic mice utilizing Tol2 transposase activity. The Tol2 transposon system not only can increase the gene integration rate, but also has the inclination to integrate foreign genes into AT rich regions.
Biocytogen incorporates a bioinformatics approach to minimize off-target activity by searching the target genome for regions of similar sequence identity to the sgRNAs. Therefore, only sgRNAs with high specificity and high activity are chosen. As part of our quality control measures, we can perform 2 rounds of PCR to amplify the genomic regions around potential off-target sites, followed by sequencing to look for off-target events. We have also integrated Southern blot in our workflow as an important quality control step for generating knock-in and conditional knockout animal models to detect any potential random insertions. A dedicated project manager will be assigned to your project once it is initiated, they will provide monthly updates on the status of your model and are available to be reached at any time.
How Our Gene Editing Services Work Biocytogen incorporates a bioinformatics approach to minimize off-target activity by searching the target genome for regions of similar sequence identity to the sgRNAs. Therefore, only sgRNAs with high specificity and high activity are chosen. As part of our quality control measures, we can perform 2 rounds of PCR to amplify the genomic regions around potential off-target sites, followed by sequencing to look for off-target events. We have also integrated Southern blot in our workflow as an important quality control step for generating knock-in and conditional knockout animal models to detect any potential random insertions. A dedicated project manager will be assigned to your project once it is initiated, they will provide monthly updates on the status of your model and are available to be reached at any time.
For more detailed information about our gene editing service technologies, Download Gene Editing Brochure.
The Flvc2GFP allele was generated by homologous recombination in embryonic stem cells. Two sets of inverted loxP sites (one regular set and one mutated set) were introduced in order to remove the second exon and flip eGFP in-frame upon Cre recombination. Validation of the cloned construct by Southern blot was achieved using two probes and two restriction enzymes.
