gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid Conditional Gene Expression Vector (LoxP-Stop-LoxP)
This regular plasmid gene expression vector system utilizes the LoxP-Stop-LoxP (LSL) cassette to achieve Cre-mediated conditional activation of gene expression in mammalian cells and animals. The LSL cassette comprises a LoxP-flanked (aka floxed) triple repeat of the SV40 polyadenylation sequence. The user-selected promoter is placed upstream of the cassette while the user’s gene of interest is placed downstream of it. In the absence of Cre recombinase, the cassette completely blocks transcription of the gene of interest. When Cre is introduced into cells carrying this vector, the cassette is excised, allowing the user-selected promoter to drive the transcription of the gene of interest.
While this vector system can be used in tissue culture cells, it is particular suitable for the generation of transgenic animals. When a transgenic animal carrying such a vector is crossed to an animal carrying a tissue-specific Cre transgene, the progeny animals carrying both types of transgenes would turn on the gene of interest specifically in cells where the tissue-specific Cre is expressed.
For using this vector system in cell culture, antibiotic or fluorescence based markers can be added to the vector to allow selection or visualization of transfected cells, including the isolation of cells that have permanently integrated the vector in the genome.
For further information about this vector system and Cre-mediated recombination, please refer to the papers below.
|Mol Biotechnol. 16:151 (2000)||Overview of vector design for mammalian gene expression|
|EMBO J. 12:2539 (1993)||Transcription blocker prevent transcriptional interference|
|J Biol Chem. 259:1509-14 (1984)||Purification and properties of the Cre recombinase protein|
|Genesis. 26:99-109. (2000)||Review of the Cre/LoxP recombination system|
This vector is designed for Cre-mediated conditional gene expression in mammalian cells and animals. Expression of the gene of interest is initially silent, but can be permanently activated by coexpression of Cre recombinase, which will excise a 3x SV40 polyadenylation sequence upstream of the gene of interest. After treatment with Cre, expression of the gene of interest is under the control of the user-selected promoter.
Stable gene activation: Treatment with Cre recombinase will permanently remove the 3x SV40 polyadenylation sequence which blocks downstream transcription. This will allow transcription of the gene of interest, driven by the promoter chosen by the user.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.
Very large cargo space: Our vector can accommodate ~30 kb of total DNA. The plasmid backbone only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.
High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.
Suitability for in vivo applications: While this vector system can be used in tissue culture cells, it is particularly suitable for the generation of transgenic animals for the purpose of Cre-mediated conditional gene expression.
Non-integration of vector DNA: When used in cell culture, plasmid DNA generally integrates into the host genome at only a very low frequency (one per 102 to 106 cells depending on cell type). Drug resistance or fluorescence markers incorporated into the plasmid can be used to isolate cells stably integrating the plasmid by drug selection or cell sorting after extended culture.
Limited cell type range: The efficiency of plasmid delivery in cell culture can vary greatly from cell type to cell type, and often requires optimization. Primary cells are often harder to transfect than immortalized cell lines, and some cell types are notoriously difficult to transfect.
Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.
Promoter: The promoter that will drive expression of your gene of interest after treatment with Cre recombinase.
LoxP: Recombination site for Cre recombinase. When Cre is present the region flanked by the two LoxP sites will be excised.
3x SV40 pA: Three repeats of the simian virus 40 late polyadenylation signal. This terminates transcription from the upstream promoter, preventing expression of the downstream gene of interest.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of the downstream marker gene.
Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
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.