Knock-In Vector Construction

Gene knock-in (KI) refers to the precise insertion of an exogenous DNA fragment into a specific genomic locus, allowing the inserted sequence to perform its intended function. This technique can endow cells or organisms with new functionalities or restore the normal function of defective genes. It is widely applied in studying gene function and regulatory mechanisms, constructing disease models, and advancing gene therapy.

Service Details

Service Types Fluorescent Protein Knock-in Vector / Tag Protein Knock-in Vector / Specific DNA Fragment Knock-in Vector
Delivery Standards 1.Plasmid map
2.Plasmid sequencing results
3.Plasmid amplification instructions
4.Plasmids (sgRNA-Cas9 editing plasmid and Donor plasmid)
Timeline/Pricing   Consult online for details
EDITGENE has developed an innovative and highly efficient gene knock-in technology, offering gene knock-in vector construction services. Utilizing an upgraded CRISPR/Cas9 system that includes sgRNA-Cas9 plasmids and Donor plasmids, we have optimized sgRNA and homology arm design strategies. Through homology-directed repair (HDR) and other mechanisms, the target gene sequence is inserted precisely at the designated knock-in site.

Service Advantages

Long Insert Fragments
Our editing vectors enable the insertion of DNA fragments up to 5 kb in length.
 
High-Activity Cas9 Plasmids
Utilizing highly active Cas9 plasmids to efficiently cleave genomic DNA, increasing overall editing efficiency.
Optimized sgRNA
Our upgraded sgRNA design algorithm ensures high-efficiency sgRNA for precise and effective gene editing.
Experienced Team
Our skilled and experienced technical team can design tailored knock-in vectors according to client requirements.

Service Types

Customized gene knock-in vectors can be designed and constructed based on client requirements and specific gene characteristics.
Fluorescent Protein Knock-in Vectors EGFP, Luc, mCherry, etc.
Tag Protein Knock-in Vectors Flag, HA, Myc, HiBiT, etc.
Vectors for Inserting Specific DNA Fragments into Target Genomic Loci /
Vectors for Inserting Specific DNA Fragments into Genomic Safe Harbor Sites /

Plasmid Map

 
pCRISPR Donor Plasmid Map
 
 
 
pSpCas9 Plasmid Map

Advantage and Characteristic

Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic

Genetic Reference Book

Insertion of tdTomato Downstream of the Ins2 Promoter in C57BL/6J Mice
Article Title: CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets

Using CRISPR/Cas9 gene editing technology, researchers constructed a transgenic mouse model to express a specific fluorescent protein in pancreatic β-cells. In C57BL/6J mice, the tdTomato transgene was inserted downstream of the Ins2 promoter. The researchers designed an Ins2-specific single-guide RNA targeting exon 2 for the CRISPR/Cas9 system and constructed a donor vector. The Cas9, single-guide RNA, and donor vector were then injected into mouse fertilized eggs in vitro, and these fertilized eggs were implanted into pseudopregnant mice. Heterozygous mating produced homozygous mice, and genotype identification, in vivo imaging, and frozen sections confirmed the knock-in effect. Six F0 mice and stably inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed no changes in the Ins2 exon compared to the control group; only the base sequence of tdTomato was added without base mutations. However, no expression of red fluorescent protein (RFP) was observed in vivo imaging and frozen sections, indicating low expression of the tdTomato protein and fluorescence intensity not reaching the detection threshold. In CRISPR/Cas9 technology, the IRES-linked foreign fragment may affect the transcription levels of the upstream gene, leading to low expression levels of the downstream gene and affecting the insertion effect.

FAQ

What host bacteria are used for vector construction in EDITGENE? What type of strains can customers use to amplify plasmid vectors?
During vector amplification, Escherichia coli (E. coli) strains are typically used. The commonly used strain for most non-recombinant vectors is DH5α, which is suitable for most applications. For recombinant vectors, such as lentiviral vectors and transposon vectors, the Stbl3 strain can be used for amplification. Stbl3 is a specialized E. coli strain derived from HB101, which has a mutation in the recombinase gene recA13, effectively suppressing recombination of long fragment terminal repeat regions and reducing the likelihood of erroneous recombination.
When selecting a vector, consider the purpose of the experiment and the type of host cells. For example, plasmid vectors are commonly used for gene expression or amplification in bacteria, while viral vectors are more suitable for gene transfer in mammalian cells. Additionally, the vector's promoter, replicon, and antibiotic selection markers should be chosen based on specific requirements.
EDGENE

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