Abstract: Transgenic technology makes it possible for humans to change the trait phenotype of organisms according to their subjective wishes. The type and quality of vectors are directly related to the efficiency of subsequent transgenes. Therefore, the construction of expression vectors has become one of the key links in transgenic research. Lentivirus, also known as retrovirus, belongs to the family of retroviruses and is an RNA virus. Since this virus contains reverse transcriptase, it is called retrovirus. Lentivirus in the host cell, using RNA as a template, synthesizing cDNA under the action of self-reverse transcriptase, and then synthesizing double-stranded DNA using cDNA as a template, and cyclizing and integrating the chromosome of the host cell by viral integrase Expressed on the long term. The lentiviral vector (LV) is constructed by the lentivirus to remove part of the gene based on its genome and is constructed with the desired gene and marker of interest. Compared with other vectors, the lentiviral vector has a large capacity to carry the gene fragment. The dyeing efficiency is high, it can infect dividing cells and non-dividing cells, and the target gene can be stably expressed in the host cell for a long time and has good safety. It has become an ideal carrier for transferring the target gene. This article will focus on thelentiviral vectorconstruction and their applications in genetics.
Keywords: lentiviral vector, construction, application
u The development of lentiviral vectors
In the early days of the development of lentiviral vectors in the early 1990s, HIV-1-based lentiviral vector research was mainly used for the study of the biological characteristics of HIV and the treatment of AIDS. The expression vector is mainly constructed for target cells expressing CD4, and most of the packaging systems retain all HIV genes as a control, and a series of viral genomes such as mutation or deletions of a certain sequence are modified. The purpose was to observe the effects on viral packaging, replication, infection, etc., and to study the genome composition of lentivirus HIV-1. This laid a solid foundation for the subsequent research of lentiviral expression vectors.
In the mid-1990s, the three-plasmid system of Didier Trono of the University of Geneva and Luigi Naldini and Inder Verma of the Sack Biological Research Institute of California marked the birth of the first generation of the lentiviral vector multi-plasmid system. The system consists of a vector plasmid, a packaging plasmid, and an envelope plasmid. The vector plasmid is responsible for loading the foreign gene, deleting genes such as gag, pol, env, but retaining 5' and 3', LTR, Psi sites, and Rev Response elements, thus improving the carrier loading while ensuring its efficiency. On the basis of this, Zufferey et al designed the second generation lentiviral vector, and deleted three auxiliary genes of vif, vpr and nef on the packaging plasmid, and obtained the infection of non-dividing cells, mononuclear macrophages and nerves. Cell-capable lentiviral vector and did not affect viral yield. Since these helper genes can express virulence viral proteins, their deletion not only increases the safety of lentiviral vectors but also increases the stability of the packaging cell line. The third generation of lentiviral vectors subsequently has higher biosafety. The vector plasmid contains only the cis-acting site and does not express any HIV protein. The packaging plasmid contains only three HIV functional genes, gag, pol and rev, and all other helper genes are deleted.
u Construction of lentiviral vector
Ø The capacity and characteristics of lentiviral vectors
Due to the restriction of the size of the inserted gene in the packaging capacity of the viral capsid, the possible loading capacity of the target gene in the vector should be considered first when constructing the vector. The length of the HIV proviral genome is about 9.8 kb, and the length of the plasmid vector can be subtracted. The maximum length of the fragment of interest is loaded, which is the biggest disadvantage of lentiviral vectors compared to micromanipulation. At the same time, the packaging systems for the 2nd and 3rd generation lentiviral vectors are not universal because the second generation vector plasmid is Tat-dependent and the third generation packaging system does not contain the Tat gene.
Ø Optimization of lentiviral vectors
The expression efficiency can be increased by inserting a regulatory sequence. For example, by inserting the woodchuck hepatitis virus post-transcriptional regulatory element into the three untranslated region of the target gene, the expression level of the target gene can be increased by 5 to 8 times.
Ø Double gene transfer vector design
In the transgenic research, the strategy of double gene co-transfer is often used. The most common method for constructing lentiviral double gene transfer vector is to design bicistronics using internal ribosome entry site (IRES). The disadvantage of this strategy is downstream. The gene expression is low, and even the expression of downstream genes is not detected at all, and the use of two transcription units in tandem on the lentiviral vector effectively overcomes this shortcoming, enabling the downstream gene to be efficiently expressed.
u Application of lentiviral vector
Treating HIV infection
With the surge in AIDS patients worldwide, the way to treat AIDS as soon as possible has become the most important issue. Lentiviral vectors have many advantages in the treatment of HIV-1 infection. First, some lentiviral vectors from HIV-1 and HIV-2 compete with viral RNA for packaging and competition for viral proteins for viral replication, inhibiting their replication, thereby controlling HIV infection. Second, lentiviral vectors can stably transfect dividing cells and non-dividing cells, especially those associated with HIV-1 replication and immune recovery, such as dendritic cells, T cells, and the like. Third, lentiviral vectors can be designed to express genes with therapeutic anti-HIV-1, specifically controlling the various stages of viral replication.
Treatment of tumor
For a long time, the treatment of tumors has been based on surgery, radiotherapy and drugs. Tumor gene therapy is a new treatment method after these traditional treatment methods. The lentiviral vector can safely and effectively implant the tumor gene into the human body for long-term effective expression. The use of lentiviral vectors increases the sensitivity of tumor cells to traditional chemotherapeutic drugs. RNAi technology is also used in tumor gene therapy. The occurrence of cancer is mainly due to the mutation and abnormal activation of proto-oncogenes. Lentivirus can specifically reduce the expression of RNAi against proto-oncogenes in various cells, thereby inhibiting canceration. Cell proliferation; shRNA capable of efficiently transmitting tumor-associated genes, silencing after transcription, inhibiting the expression of the target gene, reducing its promotion in tumor development and tumor invasion, thereby
playing a therapeutic role.
Treatment of transplant rejection
In recent years, organ transplantation has been widely used to treat severe organ failure, but how to reduce transplant rejection and graft-versus-host disease (GVHD) after organ transplantation has become the most urgent problem to be solved. Activation, differentiation and proliferation of T lymphocytes play a key role in the immune response to transplantation. Genetic modification of T lymphocytes to reduce rejection and GVHD is one of the important directions in the field of transplantation. Li Zhenyu et al. observed the expression of the lentiviral vector in mouse lymphocytes by constructing a lentiviral three-plasmid system containing the green fluorescent protein gene, and found that the lentiviral vector has high infection efficiency on mouse T lymphocytes and can be rapidly stabilized. Transfer foreign genes to T cells.
References
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