How do I know what my target gene should be. And how do I select a target gene?

Multiplicity of Infection (MOI) is a term used to describe the ratio of infectious agents, such as viruses or bacteria, to the target cells in a given experiment or therapeutic application. This concept is important in virology, microbiology, and gene therapy, as it helps determine the optimal conditions for infecting cells or delivering genes. In essence, MOI represents how many infectious particles are needed to infect a specific number of cells. Researchers aim to achieve an efficient infection rate, using as few agents as possible while ensuring that the target cells are adequately infected. In gene therapy, MOI is crucial for determining how effectively genes can be delivered to cells without wasting resources or overexposing cells to the infectious agent. To calculate MOI, the formula is straightforward: MOI = (Number of Infectious Particles) / (Number of Target Cells) For instance, if you have 1 million target cells and 10 million viral particles, the MOI would be 10. This means that, on average, each cell would be exposed to 10 virus particles. When determining the appropriate MOI for a given experiment, several factors need to be considered: 1. Cell Type and Health: Different types of cells may have varying levels of susceptibility to infection, and their health can impact how effectively they can be infected by the agent. Some cells may require a higher MOI for efficient infection. 2. Virus Characteristics: The type of virus or infectious agent being used also affects the MOI. Some viruses infect cells more efficiently than others, which means the MOI will need to be adjusted accordingly. 3. Experiment Objective: The purpose of the experiment plays a role in determining the ideal MOI. In cases where it's essential to infect all cells, a higher MOI may be necessary. Alternatively, a lower MOI might be used to infect fewer cells or to avoid overwhelming the cells with an excessive viral load. 4. Infectivity: Not all viral particles may be successful in infecting a cell. The efficiency of the virus at infecting cells should be considered when calculating the MOI, as this can influence how many particles are needed to achieve the desired infection rate. In practice, different levels of MOI have distinct uses. A high MOI (e.g., 10 or more) ensures that nearly every cell is infected by multiple infectious particles, which is useful in experiments where complete infection is the goal. Conversely, a low MOI (e.g., 1 or less) may be used when researchers want to limit the number of infected cells, such as in studies where they need to observe the effects of single-infection events or reduce toxicity in gene therapy applications. In summary, MOI is a key factor in controlling the efficiency and outcome of experiments involving infection or gene delivery. By carefully calculating and adjusting the MOI, researchers can ensure that the cells are infected at the desired rate while minimizing the potential for unwanted side effects.

The recombinant lentivirus system has become a crucial tool in molecular biology and gene therapy due to its efficiency in delivering genetic material into a wide range of cells, including both dividing and non-dividing cells. Lentiviruses, a subclass of retroviruses, have the unique ability to integrate their RNA genome into the host cell's DNA. This property has been harnessed for various applications in gene therapy and research. The mechanism behind the recombinant lentivirus system involves modifying the virus to remove its pathogenic genes and replacing them with a gene of interest. This enables the virus to deliver the therapeutic gene into target cells without causing disease. Once the recombinant lentivirus enters the target cells, it releases its RNA genome, which is then reverse-transcribed into DNA. This viral DNA is integrated into the host's genome, resulting in long-term expression of the introduced gene. This integration feature is a significant advantage over other gene delivery methods, such as plasmid-based systems, where the genetic material may not be as stably maintained in the host cells. Recombinant lentiviruses have found widespread use in gene therapy, both in vivo and ex vivo. In ex vivo gene therapy, cells are taken from a patient, modified with a recombinant lentivirus, and then reintroduced into the patient to treat specific conditions. This approach has been used to treat genetic disorders, such as sickle cell anemia, by modifying hematopoietic stem cells to express a corrected version of the hemoglobin gene. In vivo gene therapy, however, involves directly administering recombinant lentivirus into the patient's body. While this approach holds great promise, it faces challenges related to delivery efficiency, immune responses, and ensuring the stable integration and expression of the therapeutic gene. One of the main advantages of recombinant lentiviruses is their ability to integrate their genetic material into the host cell's genome, providing stable, long-term expression of the introduced gene. Additionally, lentiviruses have a broad cell tropism, meaning they can efficiently infect both dividing and non-dividing cells, making them ideal for gene transfer into a variety of cell types. Lentiviruses also generally induce a less potent immune response compared to other viral vectors, such as adenoviruses, which is crucial for ensuring long-term gene expression. Furthermore, lentiviral vectors can carry relatively large genetic inserts, making them suitable for delivering therapeutic genes or gene cassettes that require substantial genetic sequences. However, there are challenges associated with recombinant lentiviruses. Immune responses, while generally less potent than those triggered by other viral vectors, can still occur, especially with repeated administration. Managing these immune responses is a significant area of ongoing research. Another challenge is the risk of insertional mutagenesis, where the integrated viral DNA disrupts the function of host genes, potentially leading to unwanted effects such as oncogenesis. To mitigate this risk, research is focused on improving vector design and enhancing the specificity of integration sites. Additionally, the production of recombinant lentiviruses for clinical applications can be complex and costly. Improvements in manufacturing processes are needed to make lentiviral-based therapies more accessible and affordable. Looking ahead, the recombinant lentivirus system continues to hold great potential for gene therapy and genetic research. The development of gene-editing technologies like CRISPR-Cas9 could further enhance the efficiency and precision of lentiviral delivery. Advances in the modification of the lentivirus capsid and other components could improve targeting specificity, reduce immune reactions, and minimize insertional mutagenesis risks. With these advancements, recombinant lentiviruses could play a crucial role in developing personalized gene therapies for various genetic and acquired diseases. In summary, the recombinant lentivirus system is a powerful and versatile tool in molecular biology and gene therapy. While there are challenges to overcome, ongoing research is expected to refine and expand its applications, potentially transforming the treatment of genetic diseases, cancer, and other medical conditions.

I'd say the music is too loud. I couldn't complete the video.

can you please make a video how to use CAZy, where nobody have done video till now

How do I see what kind of DNA topology I'm working with?

very helpful

excellent

very helpful

Who is watching in 2025!!!!

Amasing 🎉

I have a injection on my brain

Masterfully explained

Depends on what pcr as for taqman assays, you can easily go 15-25bp

While checking the hTERT expression in immortalized cells by qPCR, do I need a control lentiviral vector without hTERT gene expression? Please reply. Thanks.

so good

interesting😍

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파이로시퀀싱 : 한종류 염기 때려넣고 반응시킴- 만약 반응이 일어나면 발생되는 빛 관찰로 어떤 종류의 염기가 들어갔나 측정, 워싱이 제대로 안되어 잡음 커짐 합성 : 4종류 염기 때려넣고 반응시킨 뒤 워싱 - 이후 현광표지 떨어뜨려 각 염기마다 첨가시 다르게 나타나는 현광을 측정하여 어떤 염기가 잔류하는지 즉 합성되어 들어갔는지 측정, 워싱 잘 안되면 안붙어있는 염기들의 빛까지 같이 측정됨. 라이게이션 : 합성과 비슷 but cg, ac 등등 두개의 염기로 측정하며 뭉터기가 붙어서 두개읽고 3칸 띄우고 다시 2대 읽고 이럼. 이후 프라이머 한 염기씩 밀려서 만들고 이를 반복해 측정하여 빈칸 채우고 정확도 올림, 시퀀싱 길이 짧음 반도체 : 파이로와 비슷하게 한종류 염기 넣고 반응 되나 안되나 확인 but 이는 합성시 나타나는 수소이온 측정하는 것으로 별도의 표지 필요없어 더 쌈.

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Well explained ❤

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Nice informative video. Thanks.

Even if you silence the tumor suppressor genes, wouldn't the finite length of the telomeres still limit the immortalization if you use only that method?

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You should have used an example to buttress your points

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Could you differentiate between the tranfer plasmid with and the envelope plasmid? For example, if I want spike proteins on the pseudoviruses , to which plasmid( transfer plasmid or envelope plasmid ) should I clone the sequence for spike protein?

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