Based on the principle and choice of different gene regulation methods of viral vectors

1. What aspects of virus vectors can
genetic
operations?
of gene function mainly involves functional gain and function loss, functional gain mainly includes the over-expression of external sources, endogenal activation, functional loss mainly includes gene interference and knock-out. With viral vectors, gene over-expression, interference, knock-out, and endogenal activation can be performed, including encoded and non-encoded genes.
2. Principles of different gene regulation
exoded over-expression:
gene function studies often need to increase the expression of the intended gene to observe esoteric changes. Over-expression is the process of mass expression of the destination gene in the host cell, the basic principle of which is to build the purpose gene into the tool carrier, and import the gene into the cell to make the gene to be transscribed and translated in large numbers, so as to realize the over-expression of the gene product.
The use of viral vectors for gene over-expression is a mature technology, researchers only need to choose the right initiator, fluorescence, labeling and resistance to build the right vector, packaged as a virus, infected cells, can achieve gene over-expression operation.
note: Before the gene over-expression, the first need for the intended cells to carry out background expression testing, low background expression, can be expressed, if the background expression is too high to consider the same kind of other cells or genetic interference operation.
genetic interference:
RNA interference (RNAi) is a phenomenon that inhibits the expression of specific genes in normal organisms, and when two-stranded RNA (dsRNA) of the same origin as endogenic mRNA coding regions is imported into cells, the mRNA degrades and causes gene expression to be silenced.
the antonym chain of small molecules that interfere with RNA (siRNA) and a variety of
nucleic acids
enzymes form a silent complex (RISC), which binds and cuts mRNA to mediate the process of RNA interference.
RNAi action mechanism
Note: Before genetic interference, the first need for the purpose cell background expression test, the background expression is high, can be interfered with, if the background expression is low, then consider changing the same other cells or genetic over-expression operation.Gene Knock-out
:
CRISPR/Cas9 is a technique that allows precise editing of specific site points in the
genome
, based on the principle that the nucleic acid intrachease Cas9 protein passes through guided RNA (gRNA) Identify specific genomic bits and cut double-stranded
DNA
, resulting in DNA double-stranded fractures, and cells repair cutting bits using non-iso-end connections (NHEJ) or iso-recombination (HR) for DNA horizontal gene knock-out or precise editing.
In gene knockout, Cas9 protein under the guidance of gRNA, the specific DNA bits are cut to produce double-stranded fractures, and then the cells through non-isocent end connections to repair the DNA, in the process, will randomly cause the loss or increase of the base, the gene code
mutation
, resulting in the removal of the encoded gene.
: For some gene expression of protein function is relatively strong, interference is not enough to produce esoteric, consider using CRISPR/Cas9 technology, genome-level knock-out for gene function research.endogenic activation/inhibition:
through the human mutation Cas9 RuvC1, HNH two shearing points, Cas9 protein cutting capacity loss into dCas9 protein, dCas9 and sgRNA complex can be combined in a specific location in the DNA DNA On this basis, the researchers connected some transcription control elements at the rear of the compound, such as transcription-activated VP64, VP16, transcription-suppressed KRAB, etc., while anchoring the compound in the upper reaches of the gene transcription starting region, to achieve transcription activation or transcription suppression of specific genes.
dCas9 technology principle
3. Differences and choices between exoded over-expression and endo-origin activation
4. The differences and choices between gene interference and gene knock-off