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Custom HR Vector Services
PrecisionX™ HR Targeting Vectors
Recent advances in tools available for precise genome engineering of target cells have revolutionized the field of biology. Transcriptional activator-like effector nucleases (TALENs), first described in the plant pathogen Xanthomonas sp., have shown that researchers can efficiently target any genomic DNA sequence using a pair of custom TALEN proteins whose DNA-binding modules can recognize individual DNA nucleotides based on an elegant amino acid cipher (Boch et al. 2009). Based on the explosion of recent publications that have successfully demonstrated the utility of the TALEN platform in many eukaryotic model organisms ranging from zebrafish to humans, it has quickly established itself as the de facto platform for genome engineering.
The Cas9/CRISPR technology is based on a bacterial system for combating invading viral and nucleic acid sequences. Originally discovered in the pathogenic bacterium Streptococcus pyogenes, this system uses an unique series of short RNAs (collectively termed as "guide RNA") to specifically target a complementary DNA sequence, and upon binding, leads to recruitment of an endonuclease called Cas9 to specifically induce a double-stranded break (DSB) in the DNA sequence targeted by the guide RNA (Bhaya et al. 2011, Jinek et al. 2012). Similar to TALENs, the introduction of DSBs in the DNA leads to recruitment of the cellular repair machinery to drive non-homologous end-joining (NHEJ) processes in absence of homologous sequences, whose error-prone nature often leads to formation of indels in the targeted DNA sequences.
However, in order to fully leverage the powerful nature of these genome engineering platforms, there needs to be a set of ancillary tools that will allow detection of cells that have been modified by TALEN or Cas9 systems. The majority of genome editing events affect only a percentage of the total population of transfected cells, estimated at 1-80% for mono- or bi-allelic modifications depending on the platform, the cell type, and the DNA target of interest. Due to the wide variation in activity, performing downstream phenotypic assays in a background of wild-type cells is challenging, especially if the phenotype in question is subtle or difficult to discern. Therefore, tools in the form of donor or targeting vectors that contain 1) fluorescent or antibiotic selection markers and 2) a gene fragment of interest to knock-in, knock-out, or correct a wild-type sequence will be extremely useful for obtaining a homogeneous population of cells whose genomes have been successfully targeted. SBI has generated a suite of homologous recombination targeting vectors suitable for the purpose of engineering both protein coding and non-coding genes including microRNAs and LncRNAs genomic loci.
Have SBI design and build a custom HR vector tailored for targeting a genomic locus of your choice
Choose from a variety of HR vector formats that best suit your goals
GFP fusion vectors
The HR120PA-1 targeting vector is designed to direct the in-frame fusion of GFP to the 3' end of your gene of choice. The 5' homology arm/GFP fusion segment is created using seamless fusion cloning technologies (e.g. SBI's Cold Fusion Kit) at the provided EcoRI site. The 3' homology arm can be cloned into the MCS using Cold Fusion cloning or traditional cloning using the provided restriction sites. An example of utilizing the HR120PA-1 vector to generate a C-terminal GFP fusion construct is illustrated above. Additionally, the EF1a-RFP-T2A-Puro cassette can be excised by transient expression of Cre recombinase, leaving only the GFP fusion protein and a single LoxP site.
The HR130PA-1 vector is designed to direct the T2A-peptide/GFP linkage to the 3' end of your gene of choice. This vector can be used in cases where a GFP fusion in-frame with the target gene may affect the function of the endogenous gene of interest. The 5' homology arm/T2A-GFP linkage segment is created using seamless fusion cloning technologies (e.g. SBI's Cold Fusion Kit) at the provided EcoRI site. The 3' homology arm can be cloned into the MCS using Cold Fusion or by traditional cloning using the provided restriction sites. The EF1a-RFP-T2A-Puro cassette can be excised by expression of Cre recombinase, leaving only the GOI-T2A-GFP construct and a single LoxP site.
Gene tagging with dual GFP and Luciferase markers
The HR150PA-1 vector is designed to direct the in-frame fusion of GFP-T2A-Luciferase to the 3' end of your gene of your choice. The gene of interest will be fused to GFP and will express luciferase as a separate protein for applications where quantitative measurement of endogenous gene expression is desired. The 5' homology arm/GFP-T2A-Luciferase fusion segment is created using seamless fusion cloning technologies (e.g. SBI's Cold Fusion Kit) at the provided EcoRI site. The 3' homology arm can be cloned into the MCS using Cold Fusion or by traditional cloning using the provided restriction sites. The EF1a-RFP-T2A-Puro cassette can be excised by expression of Cre recombinase, leaving only the GFP Fusion-T2A-luciferase construct and a single LoxP site.
IRES-GFP co-expression vectors
The HR180PA-1 vector is designed for coupled expression of the gene of interest to an eGFP marker by the use of an IRES element, which aids in translation of the eGFP protein. This vector is especially useful for monitoring expression of non-coding transcripts (e.g. microRNA, lincRNAs) that are targeted by TALEN or Cas9 systems for functional studies. The 5' homology arm can be cloned into the vector by traditional restriction enzyme cloning or by fusion cloning methods to the 3' end of your gene of your choice using the provided restriction sites directly upstream of the IRES element. The 3' homology arm can be cloned into MCS2 using the same cloning methods. Since the IRES controls the expression of the eGFP, the gene of interest does not have to be in-frame with eGFP and in cases of cDNA inserts, the presence of a stop codon in the sequence does not affect eGFP expression. The EF1a-RFP- T2A-Puro cassette can be excised by expression of Cre recombinase, leaving only the GOI-IRES-eGFP construct and a single LoxP site.