Binary control system:Split gal4(p65AD and gal4DBD)
Individual enhancers drive the expression of either GAL4's DNA-binding domain (DBD) or an activation domain (AD) joined to a leucine-zipper dimerization domain. When expressed individually, each half is insufficient to activate transcription of a UAS reporter. When both AD and DBD are present in the same cell, they combine to make a functional transcription factor that can bind to tandem arrays of GAL4’s cognate UAS DNA sequence and activate transcription.
"Q system consists of a transcriptional activator QF, QS(an inhibitor of QF), and a QF binding site called QUAS (upstream activation sequence of QF). QF is a transcriptional activator,and it binds to the sequence of Quas after expression and activates the expression of downstream target genes. QS is a QF repressor that effectively blocks QF-mediated transcriptional activation. The GeneSwitch (GS) is a modified Gal4/UAS system, whereby transgene expression is induced in Drosophila by adding the drug RU486 to food. The GS system is routinely used in Drosophila aging and behavioral studies to avoid confounding effects related to genetic background mutations."
请An enhancer trap is a recombinant enhancer trap that connects a reporter gene to a sophisticated promoter that does not initiate transcription on its own and requires the help of enhancers inserted into the cell genome to transcribe.If the reported gene is expressed, the presence of an enhancer or a gene near the insertion site can be inferred.
Cas9, located near the CRISPR site, is a double-stranded DNA nuclease that guides endonuclease to cleave at the target gene through a specific RNA sequence (GRNA for Cas9). It does not need to form a dimer to function in order to cause the target gene to produce DSB (double stranded gap).
The Phic31 integrase gene was integrated into the drosophila genome by transgenic method, and the embryo expressed itself to provide the integrase. Attp Drosophila stock is a Drosophila stock with attP locus constructed by transposon transgenic technology (such as transposons such as P factor and piggyBac).
The PHIC31 (ΦC31) integrase system allows site-specific integration of transgenes. In addition, many ATTP sites are characterized by location effects. Phage PHIC31 integrase is a recombinase that mediates sequence-specific recombination between ATTB and ATTP at two attachment sites.
The transposons including piggybac,minos or p-element can be used to carry some activating elements that activate the downstream genes of the insertion position, or express the genes contained in the transposon to construct a drosophila stock with specific functions. The same gene may be inserted at different locations, resulting in different knockout stocks of the same gene.
Transposase is an enzyme that performs transposable function. It is usually encoded by transposons and recognizes specific sequences at both ends of transposons. The transposons can be separated from adjacent sequences and inserted into new DNA target sites without homology requirements.
CRISPR-Cas9 is a powerful tool for sequence-specific genome editing.The CAS protein cuts genomic DNA at sites complementary to single guide RNAs.Insertion and deletion (indels) are usually achieved after the incision has been repaired.
Using CRISPR /Cas9 gene knockout technology, gRNA and Cas9 expression plasmids were designed and constructed for target genes, resulting in the functional region of target genes being knocked out, then a drosophila model without expression of this gene was obtained.
The method used CRISPR-Cas9 gene editing system to integrate the attP site on the chromosome of Drosophila melanogrosa.On this basis, the efficient insertion of foreign gene clusters into each attP site on the chromosome was achieved by the other mechanisms, and the formation of new products was realized.
The CRISPR-Cas9 gene knockin system is a double-stranded DNA cut by Cas9 endonuclease, and in the presence of a highly homologous DNA repair template, the organism initiates the HDR repair pathway and inserts a piece of exogenous DNA into the gene at a targeted point.
CRISPR implements point mutations by cleaving double stranded DNA in the presence of highly homologous DNA repair templates that insert a single nucleotide at the target point of the gene during repair.
SiRNA (small interfering RNA), a short segment double-stranded RNA molecule, is able to degrade specific mRNA by targeting homologous and complementary sequences of mRNA, thus blocking the expression of specific genes efficiently and specifically, and guides cells into a gene-missing phenotype. It is called RNAi(RNA interference).
Recombinase cre is a type I topoisomerase derived from bacteriophage P1, with a molecular weight of about 38kDa. It catalyzes site-specific recombination of DNA between loxP sites, resulting in DNA deletion and translocation. This enzyme requires no energy cofactor, and the Cre-mediated recombination quickly achieves equilibrium between the substrate and the reaction product.
The loxP (Locus of X-Overp1) site composed of two 13bp reverse repeats and an 8bp interval region is 34bp long and it is recognized by the recombinase. CRE can activate or inhibit gene expression, depending on its structure. The reverse repeat sequence is the specific recognition site of Cre recombinase, and the spacer region determines the direction of loxP site.
FLP recognition target (FRT) consists of two 13bp reverse repeats and a core sequence of 8 bp in length. It is the binding site of the FLP recombinase, and the direction of the FRT site determines the deletion or reversal of the target fragment when the system functions.