gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Plant Regular Plasmid Gene Expression Vector
Introducing regular plasmid vectors into plant cells is a widely-used procedure. Plasmid delivery can be accomplished by particle bombardment, chemical transfection, or electroporation. While other gene delivery vector systems have been developed (such as Agrobacterium binary vectors), these methods are still very commonly used. This is largely due to their technical simplicity, as well as good efficiency in a wide range of plants and cell types. A key feature of regular plasmid vectors is their transience, with only a low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).
For further information about this vector system, please refer to the papers below.
|Biolistic and other non-Agrobacterium technologies of plant transformation, In Plant Biotechnology and Agriculture. Academic Press, San Diego, 2012, Pages 117-129||Overview of plant transgenic methods using regular plasmid vectors.|
|GM Crops. 1:276 (2010)||Overview of plant transgenic methods.|
Our regular plasmid vectors are optimized for high copy number replication in E. coli and high-efficiency plant delivery and gene expression.
Technical simplicity: Delivering plasmid vectors into cells by particle bombardment, chemical transfection, or electroporation are all technically straightforward, and do not require other bacterial strains or cloning steps.
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: Regular plasmid vectors can often be introduced into cells at very high copy numbers (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.
Non-integration of vector DNA: Regular plasmid vectors mostly remain as episomal DNA, without genome integration. However, plasmid DNA can integrate permanently into the host genome at a low frequency. If a drug resistance is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection after extended culture.
Limited cell type range: The efficiency of regular plasmid delivery can vary greatly from cell type to cell type.
Non-uniformity of gene delivery: Although very high copy numbers can be achieved, this can be highly non-uniform. Some cells may carry many copies while others may carry very few, or none.
Promoter: The promoter driving your gene of interest is placed here.
Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
Nos pA: The nopaline synthase polyadenylation signal of Agrobacterium tumefaciens. This facilitates transcription termination of the upstream ORF.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in bacterial hosts.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.