gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
PiggyBac Conditional Gene Expression Vector (LoxP-Stop-LoxP)
This piggyBac 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.
This transposon-based, piggyBac vector system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed. The user-selected promoter, LSL casette, and the gene of interest are cloned into this region. When the helper and transposon plasmids are both present in target cells, the transposase produced from the helper plasmid recognizes the two TRs on the transposon, and inserts the flanked region including the two TRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment.
PiggyBac is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.
For further information about this vector system and Cre-mediated recombination, please refer to the papers below.
|Mol Cell Biochem. 354:301 (2011)||Review of piggyBac|
|Cell. 122:473 (2005)||Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice|
|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 piggyBac transposon-based 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 poly sequence which inhibits downstream transcription. This will allow transcription of the gene of interest, driven by the promoter chosen by the user.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the piggyBac transposon plasmid along with the helper plasmid can deliver genes carried on the transposon permanently into host cells due to the integration of the transposon into the host genome.
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 transposon vectors can accommodate ~30 kb of total DNA. The plasmid backbone and transposon-related sequences only occupies about 3 kb, leaving plenty of room to accommodate the user's sequence of interest.
Limited cell type range: The delivery of piggyBac vectors into cells relies on transfection. The efficiency of plasmid delivery 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.
5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.
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 late pA: Repeats of the simian virus 40 late polyadenylation signal. This terminates transcription, preventing expression of the downstream gene of interest prior to excision with LoxP-flanked region with Cre.
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.
rBG pA: Rabbit β-globin 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. It facilitates transcriptional termination of the upstream ORF.
3' ITR: 3' inverted terminal repeat.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.