ShRNA can efficiently express protein in stem cells, nerve cells, embryonic stem cells and other cells that are difficult to transfect. His promoters are exogenous, and the post-translation modification of the expressed protein may not be true enough, so TALEN can make up for his defects better. Firstly, the target recognition module is constructed. The nucleic acid recognition unit of TAL is 34 repeated constant amino acid sequences, in which the double amino acids at 12 and 13 have a constant correspondence with A, G, C and T, that is, ng recognizes T, HD recognizes C, NI recognizes A and NN recognizes G. In order to obtain TALE for identifying specific nucleic acid sequences, it is only necessary to continuously clone the corresponding TAL units according to DNA sequences. Because the genome size of species is different, the length of the selected specific sequence is also different. For mammals, including humans, the DNA sequence of 16-20bp is generally selected as the recognition target. Second,
Talon gene knockout
Endonuclease TALEn can be constructed by coupling Tale, which recognizes a specific DNA sequence, with endonuclease FokI. Moreover, FokI needs to form dimer to exert its activity, which greatly reduces the probability of random shearing. In practical operation, it is necessary to select two adjacent (13-22 bases apart) target sequences (generally 16-20 bases apart) in the coding region of the target gene or the junction between exon and intron to construct TAL recognition modules respectively. Two adjacent target recognition modules were fused and cloned to the N-terminal of FokI to form a eukaryotic expression vector, and a TALEN plasmid pair was obtained.
The target gene can be knocked out by transferring the TALEN plasmid pair into cells. After * * * was transformed into cells by TALEN plasmid, the expressed fusion proteins were specifically bound to the target sites. Because the FokI in the two TALEN fusion proteins are close to each other, they form dimers, exert the activity of non-specific endonucleases, cut DNA between the two target sites, form DSB (double strand breaks), and induce the mechanism of DNA damage repair. Cells can repair DNA by NHEJ (non-homozygous terminal ligation). In this repair process, a certain number of bases are more or less deleted or inserted, which leads to frame shift and the formation of target gene knockout mutants.
Transcriptional activation of TALEA
TALE, which recognizes the specific DNA sequence, is fused with the transcription factor activation domain VP64 to construct the transcription activation factor TALEA, which recognizes the specific DNA sequence on the promoter. In practice, it is necessary to select the target sequence upstream of the target gene promoter (generally 12- 18 bases) to construct the TAL recognition module. The recognition module TALE was fused and cloned to the N-terminal of VP64 to form the eukaryotic expression plasmid TALEA. The TALEN plasmid was transferred into cells, and the expressed fusion protein was combined with a specific DNA sequence near the promoter, and was combined with Pol II through the VP64 activation region, thus activating gene transcription and improving the expression of endogenous target genes.