gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
In Vitro Transcription Vector (for In Situ Hybridization)
Our in vitro transcription vectors are simple and efficient systems for RNA synthesis, applicable for a variety of research purposes. The in situ hybridization version of the in vitro transcription vector system is ideally suited for producing RNA probes (i.e. riboprobes), for use in in situ hybridization assays.
This system incorporates both a T7 and an SP6 promoter with your sequence of interest cloned between these two promoters. Under appropriate reaction conditions and in the presence of nucleotide triphosphates, either T7 bacteriophage RNA polymerase (RNAP) or SP6 RNAP can facilitate highly efficient production of riboprobes corresponding to your sequence of interest. These two promoters have opposing orientations, such that one promoter will produce a “sense” transcript while the other will produce an “antisense” transcript. Depending on the orientation of the insert and the experimental plan, the user may choose to perform in vitro transcription using either T7 RNAP, SP6 RNAP, or both. The flanking T7 and SP6 promoters can also be used as priming sites for PCR amplification or sequencing of inserted sequences. During in vitro transcription, hapten-labeled, fluorophore-labeled, or radiolabeled nucleotides may be incorporated to allow detection of probe localization for in situ hybridization assays. We recommend that you follow established in vitro transcription and in situ hybridization protocols available in published literature.
T7 and SP6 RNAPs both have certain base requirements for efficient transcription initiation which have been already incorporated into the vector design. When T7 RNAP is used, the first two nucleotides of the RNA transcript will be GG, corresponding to the 3’-end of the T7 promoter sequence, followed by your transcript sequence of interest. When SP6 RNAP is used, the first three nucleotides of the RNA transcript will be GAA, corresponding to the 3’-end of the SP6 promoter sequence, followed by your transcript sequence of interest.
Our in vitro transcription vector for in situ hybridization is engineered for run-off transcription. This means that T7 and SP6 RNAPs proceed to the end of the DNA template, and do not terminate at any specific sites within the plasmid. For this reason, the circular plasmid template should be linearized by restriction digestion prior to in vitro transcription. This vector system includes unique restriction sites for BsiWI, AgeI, and AscI next to the T7 promoter, and AvrII, XhoI, NotI, and SapI sites next to the SP6 promoter. Any of these enzymes will cut the plasmid at unique sites immediately downstream of your sequence of interest. Care should be taken that the sequence to be transcribed does not contain any restriction sites for the linearization enzyme used, as this would result in truncated mRNA transcripts. Contaminants from the digestion reaction may inhibit the subsequent in vitro transcription reaction, so purification via column or phenol:chloroform extraction following digestion is recommended. However, it is generally not necessary to purify the promoter and insert fragment away from other fragments, because only the fragments containing the promoter sequences will serve as templates for transcription.
For further information about this vector system, please refer to the papers below.
|Nucleic Acids Res. 7:1931 (1979)||Cloning and characterization of the T7 promoter|
|Nucleic Acids Res. 21:5480 (1993)||Characterization of the SP6 promoter|
|Methods. 52:322 (2010)||Methods for generating RNA probes by in vitro transcription, and their use for in situ hybridization.|
Our in vitro transcription vectors for in situ hybridization are designed to serve as highly effective templates for T7 or SP6 RNAP-mediated in vitro transcription. This vector system is optimized for high copy number replication in E. coli, efficient restriction digestion, and abundant RNA production.
High efficiency: Both T7 and SP6 RNAPs are robust and highly efficient enzymes. In vitro transcription reactions using either enzyme can produce large amounts of functional RNA.
Technical simplicity: In vitro transcription using a plasmid template is technically straightforward compared to other methods of probe synthesis.
Dual orientation: This vector system contains two promoters suitable for in vitro transcription, flanking the insert in opposite orientations. The choice of T7 or SP6, along with consideration of the orientation of the insert sequence, allows the user to synthesize either sense or anti-sense riboprobes.
Run-off transcription: Efficient in vitro transcription using this vector system requires linearization of the plasmid by restriction digestion prior to the transcription reaction.
T7 promoter: A promoter for the RNA polymerase from T7 bacteriophage. Drives high-level transcription of the downstream sequence of interest. This promoter is in the opposite orientation to the SP6 promoter, and will generate a transcript which is the reverse-complement of that produced from the SP6 promoter using the same template.
Transcribed sequence: Your DNA sequence of interest to be transcribed into RNA is placed here.
SP6 promoter: A promoter for the RNA polymerase from SP6 bacteriophage. Drives high-level transcription of the downstream sequence of interest. This promoter is in the opposite orientation to the T7 promoter, and will generate a transcript which is the reverse-complement of that produced from the T7 promoter using the same template.
BsiWI, AgeI, AscI, AvrII, XhoI, NotI, SapI: Unique restriction endonuclease sites that can be used to linearize the plasmid prior to in vitro transcription.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.