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AAV,动物实验,腺病毒
z-bo
2020-07-17
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Adeno-associated viruses belong to the Parvovirus family, which are non-enveloped single-stranded linear DNA viruses with a genome size of approximately 4.7 kb. Using adeno-associated viruses can transfer foreign genes into animal tissues and cells. It has many advantages such as high safety, low immunogenicity, wide host range and stable expression. It is widely used in animal studies.
Many adeno-associated virus serotype (12 kinds), different serotypes different affinities for different organizations, so the specific research, the following problems are often plagued by scientific researchers: what serotypes choose? How to inject? How much virus is injected? How to choose the injection site? How long is the injection tested? This article makes a simple summary of common injection sites (brain, retina, liver, heart and arteries, lungs, kidneys, muscles, intestines) and methods, hoping to provide you with a concept and ideas.
1. Brain
The commonly used serotypes of brain tissue are AAV1, AAV2, AAV5, AAV8 and AAV9, among which the common serotypes of nerve tissue are AAV2, AAV5 and AAV8. For brain tissue, AAV5 is more widely used and AAV8 is more effective (notably , In the nerve tissue of monkeys, AAV5 is more effective). From the perspective of effect, AAV8 is greater than AAV1.
Targeting different parts of brain tissue, the targeting of different AAV serotypes is different:
1) Hippocampus: AAV9 is more effective than AAV8;
2) Different cells of nerve tissue, different serotypes have different targeting: for example, the commonly used serotypes of neurons are AAV1, AAV2, AAV5 and AAV9; the commonly used serotypes of astrocytes and oligodendrocytes are AAV1 and AAV5.
Overall, AAV2 has the widest applicability among all tissues and is also reflected in brain tissues. For example, it can reach 75% of brain tissues. AAV2, AAV5, AAV8 and AAV9 are also relatively common, and should be selected carefully according to different organizations. Here are a few special cases:
1. Enteric Nervous System (ENS) is a complex network composed of nerve cells throughout the gastrointestinal digestive tract. The number of neurons is equivalent to the spinal cord (about 100 million nerve cells). This nervous system belongs to the vagus nerve in the lower part of the human trunk and is distributed between the digestive muscles in a thin layer to regulate digestion. Commonly used AAV5, AAV6, AAV8 and AAV9. AAV6 has certain targeting to enteric glial and neurons. The injection site can choose the descending colon, injection volume and titer: the virus with a titer of 1.3 × 1012 GC/ml can be injected with 5 μl, and the detection time: the expression starts 2 weeks after injection and is detected in 2-6 weeks.
2. The common serotype of brain tissue is AAV9, injection method: left ventricular injection or lateral ventricle injection, injection dose and titer: select viral solution with a titer of 0.5–4.0 × 1011 GC/ml, dilute to 100 μl, and inject Two weeks later (14d), the test started. injection
2. Retina
The commonly used serotypes of retinal tissues are AAV1, AAV2 and AAV5. Due to their special characteristics, retinal tissues have their own characteristics: the injection success rate is 20-30%, which is easy to cause retinal inflammation and damage. After injection, 1% atropine (and a small amount) Neomycin, polymyxin B sulfate and dexamethasone eye ointment are applied to the eye to prolong the pupil dilation time, reduce glandular secretion, and reduce inflammation), continuous use for 3-5 d. The commonly used injection volume is 0.5 μl, virus titer 1.5x107 GC/ml. Common injection methods: subretinal injection or vitreous injection.
However, different serotypes have different characteristics: AAV1 mainly acts on retinal epithelial cells and can be detected 2-4 weeks after injection; AAV2 mainly acts on photoreceptor cells, retinal epithelial cells and vitreous. AAV2 is widely distributed after vitreal injection, 2-4 weeks Can be detected; AAV5 can act on retinal epithelial cells and photoreceptor cells, and express AA5>AAV2 in photoreceptor cells for 2-4 weeks of detection.
3. Liver
For the liver, the main adeno-associated virus vectors are: AAV2, AAV5, AAV7 and AAV8, of which AAV7 and AAV8 are 10-100 times more efficient than AAV2. The injection site often chooses portal vein, peripheral vein and tail vein. Virus titers are generally selected to be 8×1010-2×1012 (GC/kg) (human, primate); 1×1011 GC/mouse, diluted to 50-100 μl for tail vein injection; 2 weeks after injection Can be detected.
IV. Heart and arteries
For the heart and arteries, commonly used vectors are: AAV1, AAV6, AAV8 and AAV9. Among them, AAV2, AAV5, AAV7 can also be used, but the expression is relatively slow, and the expression level will be equivalent to the expression level of AAV1 and AAV6 after 3 months. Among all serotypes, AAV8 and AAV9 were the most effective, and their expression levels reached the highest in about 2 months. Common injection sites are coronary artery, aorta, myocardium and tail vein. The virus titer injected is 1.9×1011-1012 GC/ml, diluted to 100μl-250μl (rat dose is slightly higher than mouse) injection; but for heart origin injection, 1010 GC/ml virus titer is often used, diluted Use into 20 μl. Detection time: Generally it can be detected after 2 weeks. The following picture shows some situations of injection:
V. Lungs
Commonly used adeno-associated virus vectors in the lungs are AAV1, AAV2, AAV5, AAV6, and AAV9. Due to the large distribution of sialic acid receptors on the lung surface, the AAV5 type is more commonly used; in contrast, the transduction efficiency of AAV2 and AAV6 is comparable. Common injection methods include nasal drip, nebulized inhalation, or intratracheal injection. The virus titer is 1012 GC/ml, diluted to 3 μl, and the expression can be detected 2 weeks after the injection.
VI. Kidney
Commonly used vectors for kidney are AAV2, AAV8 and AAV9. The titer of injected virus is generally selected from 1-5×1011 GC/ml, diluted to 50-200 μl for injection. Common injection sites include renal artery, renal vein and left iliac vein, and abdominal aorta. The expression can be detected after 2 weeks of injection.
Seven, muscle
Commonly used serotypes of muscle tissue are AAV2, AAV8 and AAV9. Among them, AAV8 and AAV9 are the most commonly used, as are myocardium, pancreas and adrenal gland. Skeletal muscle is divided into fast and slow muscle fibers, AAV2 often acts on slow muscle fibers; AAV6 is effective for both. For mice of different ages, the injection dose is different: the amount of virus selected by the young mice is 7×1010 -1.2×1011 GC/g; the amount of virus selected by the adult mice is 3.5 ×1011 -7×1012 GC/piece; but Low doses of AAV9 cannot reach all cardiomyocytes.