Introduction and application of lyovirus packaging system

, introduction to lentivirus packaging and its use
Lentivirus vector is based on HIV-1 (human immunodeficiency type I virus) based on the development of the
gene
treatment vector. Different from the general retrovirus vector, it has the ability to infect both dividing cells and non-dividing cells. The research of lysovirus vector is developing very fast and the research is very deep. The vector can effectively integrate the exogeneted gene into the host chromosome, thus achieving persistent expression. In terms of infection ability can effectively infect neuron cells, liver cells, cardiomyocytes, tumor cells, endotrical cells,
stem cells
and other types of cells, so as to achieve good gene therapy effect, clinical research has been carried out in the United States, the effect is very ideal, so it has broad application prospects.
virus is now widely used in studies expressing RNAi. Because some types of cell lipids have poor transsection effects and have a short half-life of siRNA transferred to cells, the inhibition of gene expression by in-body synthetic siRNA is usually short-lived, thus limiting its application. Using the strategy of building a carrier in-body that can express siRNA in advance and then transfer it to transcription of siRNA in cells not only increases the types of cells that are effectively transposed by lipids, but also inhibits gene expression as well as in-body synthetic siRNA, and can even play a long-term role in blocking gene expression in cells with long-term stable expression vectors. In the siRNA expression vectors constructed, RNA polymerase III. initiators guide RNA synthesis because RNA polymerase III. has a clear start and end sequence, and the synthetic RNA does not have polyA tails. When RNA polymerase III. encounters 4 or 5 Ts in a row, its guided transcription stops, forming 1 to 4 U at the 3' end of the transcription product. U6 and H1 RNA initiators are two RNA polymerase III. dependent initiators, characterized by the fact that the initiators' own elements are located upstream of the transcription region and are suitable for expression to 21ntRNA and to 50ntRNA stem ring structure (stem loop). In siRNA expression vectors, the justice and ananthrotic chains that make up siRNA can be tweed separately by their respective initiaters, and then the two chains complement each other to form siRNA, or the vector can directly express small hairpin RNA (small hairpin RNA), shRNA), the vector contains a sequence of stem ring structure located between the RNA polymerase III. promoter and the 4 to 5 T transcription termination site, which can then be folded into a stem ring structure with 1 to 4 U 3' protruding ends, which is further processed into siRNA within the cell. The vector is usually constructed by synthesized siRNA, the efficient siRNA method is found, and then the sequence that meets the requirements of the vector is selected and introduced into the siRNA expression vector.
lentiviral vector has a wider host range than retrovirus vectors, and lentivirus can effectively infect non-periodic and silky cells. ROPV vectors are capable of producing high-titular lysovirus that express shRNA, expressing shRNA in periodic and non-cyclical cells, stem cells, fertilized eggs, and differentiated offspring cells, enabling functional silence of specific and stable gene expression in multiple types of cells and genetically modified mice, providing the possibility for rapid and efficient study of gene function in progenitic human and animal cells
tissues
, as well as the possibility of producing animals with reduced specific gene expression.
virus expression vector contains the genetic information needed for packaging, trans-infection and stable integration. Lyovirus packaged protons provide all the auxiliary proteins needed to transprine and package RNA to recombined fake virus vectors. In order to produce high-titular virus particles, it is necessary to use the expression vector and packaging protons at the same time transposed cells, in the cell for viral packaging, packaged fake virus particles secreted to the
culture
base outside the cell, centrifugation can be directly used for the host cell infection, the destination gene into the host cell, after reverse transcription, integration into the
genome
, so as to achieve a high level of expression molecules.2. The purpose of this system is mainly to solve the following problems:
1. For some of the more difficult trans-infected cells, such as
primary cells
, stem cells, non-differentiated cells, etc., can greatly improve the efficiency of the transfer of the destination gene, and the chances of the destination gene integration into the host cell genome greatly increased, which provides a favorable way for RNAi, c
DNA
cloning and the study of reported genes.
2. Screening of stable cell
; 3. To provide high-quality viral fluid containing the gene of purpose for live animal model experiments,
In cell-related experimental operations, for some cells that are difficult or even impossible to transduce by conventional methods, virus-mediated experiments can greatly improve the transduction efficiency of genes in order to achieve efficient instantaneous expression of the end gene.3. Lentiviral vector
(Lentiviral, vector LVs) is a virus vector system based on HIV-1 virus, which can efficiently introduce the gene of destination (or RNAi) into the original cells or cell line of animals and humans. The virus vector genome is a positive-chain RNA in which the genome enters the cell and is converted into DNA by the reverse transcriptase it carries in the cell pulp, forming a pre-DNA integration complex, which is integrated into the cell genome after entering the nucleus. The integrated DNA transplues mRNA, returns to the cell pulp, expresses the target protein, or produces RNAi interference.
gene expression or RNAi interference mediated by the virvirus vector continues to be stable because the destination gene is integrated into the host cell genome and divides as the cell genome divides. In addition, lysovirus vectors can effectively infect and integrate into non-dividing cells. The above characteristics make the lyroviral vector compared with other virus vectors, such as the non-integrated adenovirus vector, the low integration rate of adenovirus vector, only the integration of the traditional retrovirus vector dividing cells, has distinct characteristics. Numerous literature studies have shown that the long-term expression of tissues or cells of the intended gene mediated by virulent vectors includes the brain, liver, muscles, retina,
hematopoietic stem cells
, bone marrow-to-bone marrow-filled stem cells, macrophages, etc.
virus vector does not express any HIV-1 protein, immunogenicity is low, there is no cellular immune response at the injection site, the body fluid immune response is also low, does not affect the virus vector's second injection.4. Construction of lyptogene vectors 1. Construction Principles
LYVs belong to the retrovirus department, but their genome structure is complex, including four auxiliary genes, vif, vpr, nef, vpu, and two regulatory genes, tat and rev, in addition to gag, pol, and env, three structural genes similar to simple retrovirus. HIV 21 is the most characteristic virus in virus, the first lyrovirus vector system is built on the basis of this virus. Lyovirus vectors are built to separate the sequences of the transverse action elements (such as packaging signals, long-end repeat sequences) and encoded trans-acting proteins in the HIV 21 genome. Carrier systems include packaging ingredients and carrier components: packaging components are constructed from the sequence required for packaging, reverse transcription, and integration of the HIV 21 genome, providing the proteins needed to produce viral particles in reverse; At the same time, it has multiple clone bits under the control of heterogeneity starters and the purpose gene inserted at this bit. To reduce the likelihood of replication-capable viruses (RCVs) being recombined by two components, replace the packaged ingredient with a 5' LTR with an immediate early initiator of cytocytovirus (CMV), replace the 3' LTR with an SV 40 polyA bit, and so on. The packaging ingredients are constructed on two protons, one expressing gag and pol and the other expressing env. Based on this principle, the likes of Naldini and Kafri have built three-plasmed expression systems.