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Applications of AAV and LV in Nervous System Research

Applications of AAV and LV in Nervous System Research

Recombination adeno-associated Virus, rAAV

Adeno-associated virus (AAV) is a member of the parvoviridae family. It is a type of icosahedral parvovirus that cannot replicate autonomously and has no envelope. Its diameter is about 20-26nm and contains 4.7kb. The left and right linear single-stranded DNA serves as the genome. The recombinant adeno-associated virus (rAAV) used in the study is a genetic vector modified on the basis of non-pathogenic wild-type AAV. Due to its diverse types, extremely low immunogenicity, high safety, with a wide range of host cells (infecting both dividing and non-dividing cells), strong spreading ability, and long time for gene expression in the body, rAAV is regarded as one of the most promising gene research and gene therapy vectors.

AAV genome structure Fig.1 AAV genome structure.

The process of rAAV infecting cells

The purified AAV virus vector can be used to infect cells. When infecting cells, AAV binds to specific receptors on the cell surface, activates intracellular signaling pathways, and then triggers AAV to enter cells through receptor-mediated endocytosis. With the assistance of endosomes and Golgi organelles, it enters the nucleus, and then the virus is lysed. The single-stranded DNA needs to be replicated into double-stranded DNA to express the target gene.

Mechanism of action of rAAV infecting cells. Fig.2 Mechanism of action of rAAV infecting cells.

Applications of rAAV in Neuroscience

rAAV vectors are widely used in nervous system research due to their low immunogenicity, wide range of host cells, strong diffusion ability, and diverse serotypes. They are commonly used to deliver fluorescent labels, calcium indicators, and physiological manipulation tools to mammalian brains. Such as genetically encoded calcium indicators (GECIs: GCaMP, jRGECO1), genetically encoded voltage indicators (Voltron, GEVIs: SomArchon), neurotransmitter probes (such as DA/Ach/NE/iGABASnFR) and optogenetic/chemical genetic elements (Such as ChR2, eNpHR, hM3Dq, hM4Di) are easily packaged and delivered via AAVs.

rAAV is an ideal vector for expressing minigene sequences. We can use rAAV to manipulate genes in cells, including gain of function and loss of function. However, gene sequences like spCas9 are too large to adapt to rAAV vectors. Larger genes are occasionally delivered as split modules through AAVs recombination in host cells or using lentiviruses.

When using rAAV vectors for gene delivery in vivo, the choice of virus injection method, injection volume, and serotype are particularly important. The most commonly used in the central nervous system include rAAV2/1, rAAV2/5, rAAV2/8, rAAV2/9, rAAV2/retro, rAAV2/PHP.eB, etc. These viral vectors have a certain affinity for different cells. Different serotypes should be selected according to the purpose of the study. Creative Biolabs has many years of AAV application experience, and our team of professionals will happily help you to deal with any issue related to AAV applications in brain research.

Applications of AAV and LV in Nervous System Research Virus titer: rAAV2/1-Cre: 1.13E+13 VG/mL
Injection volume: 60nl
Observation time: 4 weeks
Image source: Sci Adv. 2019 Feb 20;5(2): eaat3210. doi: 10.1126/sciadv.aat3210.

Applications of AAV and LV in Nervous System Research 4 Virus titer: rAAV2/8: 2.45E+12VG/mL
Injection volume: 500nl
Observation time: 6 weeks
Image source: Sci Adv.2019 Feb 20; 5(2): eaat3210. doi: 10.1126/sciadv.aat3210.

Viral vector: rAAV2/retro-syn-EGFP. rAAV2/retro is retrograde and does not cross synapses. Viral vector: rAAV2/retro-syn-EGFP. rAAV2/retro is retrograde and does not cross synapses.

In in vitro experiments, the traditional view is that the efficiency of rAAV is limited and the MOI required to infect cells is high. However, a variety of new rAAV vectors developed in recent years have provided us with some new ideas. For example, AAV of the rAAV-DJ serotype is also very effective in infecting cell lines in vitro, which provides new ideas for researchers.

rAAV2/9-hSyn-GFP infects mouse primary neurons, and the expression of GFP is observed 7 days after virus transfection rAAV2/9-hSyn-GFP infects mouse primary neurons, and the expression of GFP is observed 7 days after virus transfection.
Image source: CurrProtoc Neurosci. 2019 Apr; 87(1): e66. doi: 10.1002/cpns.66.

Lentivirus

Lentivirus is a type of viral vector modified from human immunodeficiency virus (HIV), which is a type of retrovirus. The genome of LV is RNA, which is a pseudotyped virus. LV can use reverse transcriptase to integrate foreign genes into the genome to achieve stable expression, and has the characteristics of infecting dividing and non-dividing cells.

After entering the cell, the lentiviral genome is reverse-transcribed into DNA in the cytoplasm to form a pre-DNA integration complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA and returned to the cytoplasm to express the target protein; or produce small RNA. Lentivirus-mediated gene expression or small RNA interference is continuous and stable, and it divides with the division of cell genome.

Applications of AAV and LV in Nervous System Research 7

Lentiviral vectors are mainly characterized by a wide host range and large gene capacity. In addition, since LV infection has integration characteristics, foreign genes can be effectively integrated into the host chromosome, and persistent expression can be achieved. However, LV has a certain degree of immunogenicity, and the characteristics of random integration may lead to abnormal cell function genes, resulting in the application of LV vectors in neurobiological research not as widely as rAAV vectors. Compared with adeno-associated virus, lentivirus has a smaller spreading range, but it expresses faster and has a larger gene capacity (accommodating 4kb), and can be used as a carrier for large-capacity genes.

Applications of AAV and LV in Nervous System Research 8 Injection volume: 1uL
Injection speed: 100nL/min
Observation time: 4 weeks
Image source: MolPsychiatry. 2019 Aug 6. doi: 10.1038/s41380-019-0472-7.

Applications of AAV and LV in Nervous System Research 9 Virus titer: LV: 9.98E+8TU/mL
Injection volume: 2uL
Observation time: 2 weeks
Image source: NatNeurosci. 2017 May; 20(5): 690-699. doi: 10.1038/nn.4536.

When used in vitro, lentiviral vectors can effectively infect cultured neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells and many other types of primary cells and most cell lines.

The lentiviral vector was used to infect primary rat neurons, and the GFP expression was observed after 48 hours of virus transfection with 5uL virus volume The lentiviral vector was used to infect primary rat neurons, and the GFP expression was observed after 48 hours of virus transfection with 5uL virus volume (titer: 1.2E+8TU/mL).
Image source: CurrProtoc Neurosci. 2019 Apr; 87(1): e66. doi: 10.1002/cpns.66.

Creative Biolabs has been committed to providing one-stop solutions for neuroscience research. Our services range from gene screening, gene function research to mechanism research. We provide one-stop technical services for plasmid construction, virus packaging, gene overexpression, gene interference, stable strain construction, cell function experiments, and animal model construction!

References

  1. [1] Curr Opin Virol. 2016 Dec; 21: 61-66.doi: 10.1016/j.coviro.2016.08.004.
    [2] Sci Adv. 2019 Feb 20; 5(2): eaat3210.doi: 10.1126/sciadv.aat3210.
    [3] Curr Protoc Neurosci. 2019Apr; 87(1): e66. doi: 10.1002/cpns.66.
    [4] Nat Neurosci. 2017 May; 20(5): 690-699.doi: 10.1038/nn.4536.
    [5] Mol Psychiatry. 2019 Aug 6. doi:10.1038/s41380-019-0472-7.
    [6] Curr Protoc Mouse Biol. 2018Dec; 8(4): e58. doi: 10.1002/cpmo.58.
    [7] Front Mol Neurosci. 2020 Jul 17; 13: 129.doi: 10.3389/fnmol.2020.00129.
    [8] Curr Protoc Neurosci. 2019Apr; 87(1): e67. doi: 10.1002/cpns.67.

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