Gene Regulation

Q: How to perform intratumoral injection of AAV?
A: Recommended dosage for intratumoral AAV injection based on literature is 2E+08–2E+11 vg, administered in two injections spaced 3 days apart.

Q: I want to knock down a gene, but its expression level is extremely low in regular cells, making it unsuitable for designing interference sequences. However, its expression is confirmed in neurons. Is there any workaround? 
A: Since the target gene has a low expression level, you can consider using high-sensitivity in situ hybridization techniques like RNAscope, which significantly improve the signal-to-noise ratio and help detect low-abundance transcripts in neurons.

Q: Overexpression of toxic proteins? Can I construct Retrovirus-DTA-GFP? DTA is the diphtheria toxin A subunit, which is highly cytotoxic and easily kills cells. Is it possible to package the virus?
A: It’s hard to say definitively, as toxicity may have an effect. You could try using a broad-spectrum promoter. Toxicity depends on protein expression level and duration, while virus packaging is completed within 48–72 hours.

Q: Can you customize an AAV system for overexpressing the Cyp19a1 gene, with the following requirements: cross the blood-brain barrier, activate only the cells in the CNS that naturally express Cyp19a1 (whether neurons or glia), and have no effect on cells that do not express the gene or on peripheral tissues including neurons?
A: If there is a sufficiently specific promoter or transgenic mouse model available (Cyp19a1-Cre), then yes, it can be done.

Q: When screening sgRNA, since monkeys lack suitable antibodies and Western blot validation isn’t possible, can qPCR be used instead?
A: qPCR does not reflect the interference efficiency accurately. It’s better to use mismatch enzyme digestion and sequencing for validation.

Q: What is the backbone used for RNAi viruses? Can a specific promoter be added?
A: The standard RNAi viral backbone is U6-shRNA-CMV-fluorescence. There are three product types: one for direct expression, one using mir30a for tissue-specific expression, and one that is Cre-dependent.

Q: Can you do interventions targeting DRG (dorsal root ganglion)? I know intrathecal injections target spinal cord and DRG, but is there a method for directly targeting DRG?
A: Yes, the DRG can be directly targeted via in situ injection. However, this approach presents technical challenges, especially in accurately delivering the injection into the DRG.

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