What is the best method for transfecting HEK293 with multiple plasmids in order to generate chimeric HIV virions?

03 August 2015 13 4K Report

I currently use calcium phosphate to transfect multiple plasmids into HEK293 cells in order to generated the chimeric HIV virions I use, but calcium phosphate does not work well a large percentage of the time. I was wondering if anyone knew of a better/more efficient/more consistent transfection method for virus production from HEK293 cells, especially using multiple plasmids to generate the virus.

Lea Johanna Bayer

We routinely use PEI transfection with HEK293T cells (to transfect between one and three different types of plasmids at a time) and it works very well!

Jill Muhling

As Lea said, PEI is good and very inexpensive. One of the problems with CaPO4 is that it's very pH sensitive - if you aren't already, add HEPES to your media when transfecting. I do find PEI much easier though!

Ezequiel Marron Fernandez de Velasco

Hi Peter,

As Lea and Jill pointed out, PEI is a common method for transfecting large amounts of DNA for viral production. I have copied bellow the answer I gave a while back to a very similar question in which you can find a standard protocol:

I have used PEI for transfection of HEK cells with good results (although efficiency is normally lower than with other methods). The method I used is for the packaging of AAVs and requires the cotransfection of 3 different plasmids; here is the protocol I used that you can adjust for your own experiment. Keep in mind that this is a very standard protocol followed by many Viral vector cores; you will be surprised on the amount of DNA that it uses but it works quite well for packaging of viruses. You could always adjust it slightly so that you don't need as much DNA:

1. For each 15cm plate to be transfected, add 100 ug of DNA to sterile 150mM NaCl for a final volume of 2ml. 6x15cm plates = 12ml 150mM NaCl+DNA. Mix well.

2. Add 75ul of PEI (16mg/ml) to 12ml 150mM NaCL+DNA (600µg DNA) while mixing.

3. After 10 minutes of incubation at room temperature, add 2ml solution complex dropwise to each 15cm plate containing 20ml medium with 10% FBS and without antibiotics. Mix gently by rocking the plate back and forth. Incubate the plate over night at 37oC in a 5% CO2 incubator.

4. After incubation, wash the cells with warm CMF-PBS and replace the medium with 20ml of pre-warmed growth medium.

FOR THE CELLS:

Maintain AD-293 cells in growth media without antibiotics in T-75 flasks. Split at a ratio of 1:4 to 1:6 every two days. Discard cells after 20 passages. Frequent passages and maintaining the cells at less that 60% confluency helps to ensure good transfection efficiencies.

Split cells the day before transfection and seed 15-cm dishes with 4 x 10E6 cells per dish in 20ml of growth media without antibiotics. The cells should be 40-60% confluent at the time of transfection. One T-175 flask at 60%-80% confluency should give 6 x 15-cm dishes for transfection. A typical AAV prep consist of 6x15cm plates for each prep.

Incubate cells overnight at 37oC and 5% CO2. The following afternoon carefully wash the cells with CMF-PBS and replace the medium with 20ml of DMEM-10% FBS Do not include antibiotics and be sure to pre-warm the medium and CMF-PBS to 37oC. Replace the medium on each plate individually two hours before PEI transfection.

Hope this helps,

Ezequiel

Lisa Marie Smith

I have co-transfected plasmids into HEK293T cells to generate virus-like particles using DharmaFECT Duo and it worked well. I imagine other lipofection reagents could work just as well. I have also tried electroporation with mixed results.

Peter Jesse Gaskill

Thank you all for your responses. I have tried PEI and it seems to work well with a single plasmid, but I have found when I am transfecting with multiple plasmids at different amounts of DNA for each plasmid - I need to transfect in a 1:5 ratio usually - I do not get as good of results. But I had not realized that PEI was so pH sensitive, so I will be sure to monitor pH in the future when I use it. Lea and Jill, is it possible for you to post your protocol for the transfection with multiple plasmids, perhaps I am doing something wrong that I am unaware of?

Jill Muhling

OK, this is how the PEI solution is made:

1. pH 50 ml 1 x PBS to 4.5 using HCl.

2. Add 50 mg linear, 25 kDal (both linear form and molecular weight are important) PEI

(Polysciences cat #23966-2) to 50 ml 1 x PBS, pH 4.5.

3. Place in a 75 oC. water bath

4. Vortex every 10 min until completely dissolved. (takes ~ 30 min or so)

5. Cool to room temperature.

6. Filter sterilize through a 0.45 um filter.

7. Aliquot (e.g., 1 ml) and store at -80 oC

To transfect cells (using T-75 scale as an example):

1. Plate your cells so that they're ~90% confluent on the day of transfection

2. In 225ul OptiMEM, add your transfer and packaging plasmids to a total of 25ug of DNA (using second or third gen plasmids, whatever you usually do)

3. Add 100ul of your PEI solution at room temperature. That is, you need 4ul of PEI for every 1ug of DNA. Incubate at room temperature for at least 20 minutes (it can be longer, complexes are quite stable)

4. Add the solution to the culture media, swirl to mix. There's no need to change the media as the 293Ts seem to tolerate PEI well.

5. Check transfection efficiency 24h post infection. In my experience, you won't see as many fluorescent cells as you would with CaPO4 BUT the lentivirus titre will still be good. I have observed that expression from a PEI transfection takes a little longer than for Lipofectamine or CaPO4 but I've never measured that properly so I could be wrong.

Hope that helps!

Peter Jesse Gaskill

Thats great, thanks so much.

Panduranga Rao

We use two plasmid transfection for recombinant adenovirus generation and PEI is much better than CaPO4 method.

Yanhg Yu

Here are the virus protocol of AAV, lentivirus and adenovirus, hope they can help you with the following step.

Antonio di stasi

I found lipoD293 to be effective and consistent for lentiviral transfection, including vectors with inserts of large size. it is a liposomal formulation with a NLS signal.

My DNA ratios for lenti transfection are 10 ug of template, 8 ug of packaging plasmid and 2.1 ug of VSVG.

May increase the packaging for inserts of larger size.

For the CaPO4 transfection I am trying the protocol from ABM, as they advocate giving better titers for retroviral supernatant.

They incubate Calcium and DNA in water for 5 minutes (50 mcl calcium 2M in 500 mcl for a 100 mm dish).

My DNA ratios for retro transfection are 10 ug of template, 8 ug of packaging plasmid and 4 ug of VSVG.

May increase the packaging for inserts of larger size.

I will try also a single plasmid with gagpol VSVG. I used to use the RDF envelope, but I prefer VSVG for better stability and possibility of concentration by centrifugation (sucrose gradient requiring only 10K rcf).

I finger click it well, because you can see the calcium as not being completely mixed after pipetting only. Then, they add the HBS dropwise (slowly). I mixed for 30 seconds and add directly to the 293T.

In this experiment, I also added chloroquine 4 hrs before transfection.

I changed the medium after 14 hours. One paper mentioned optimal results when changing the medium after 6 hours. No need to change the medium before transfection.

I am currently growing my 293T in 5% FBS and not 10%.

Antonio di stasi

I tried linear PEI 25K, PEImax and a PEI25K-PEG1K-PEI.

I am not sure why especially the PEIPEGPEI is not consistent: some time I observe no transfection.

When it happens, the PEIPEGPEI is very bright.

I found that the acidified lPEI (dissolved in HCL 0.2N) gives also a brighter transfection.

However, it is not consistent and I was not able to pinpoint why.

https://pubmed.ncbi.nlm.nih.gov/20309632/

Antonio di stasi

Cells may be too confluent. Try reverse transfection with CaPo

Yu Wa

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You

How to check if a virus is DNA virus or RNA virus and whether the virus have single stranded nucleic acid or double? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Reader,

Determining whether a virus is a DNA virus or an RNA virus, and whether its nucleic acid is single-stranded or double-stranded, involves a series of scientific procedures that are grounded in molecular biology. The process is meticulous and requires a combination of laboratory techniques to accurately characterize the viral genome. Here, we outline the steps and methodologies involved in such determinations:

Extraction of Viral Nucleic Acid: The initial step involves the isolation of the virus from an infected host or sample. Following this, viral nucleic acid is extracted using various chemical or enzymatic methods designed to separate DNA or RNA from proteins and other cellular components.

Nucleic Acid Amplification and Detection:Polymerase Chain Reaction (PCR) for DNA Viruses: PCR is a technique used to amplify specific DNA sequences. If the virus is a DNA virus, PCR can be directly applied using primers specific to conserved viral sequences. The presence of amplified products indicates a DNA virus. Reverse Transcription PCR (RT-PCR) for RNA Viruses: For RNA viruses, reverse transcription is performed to convert RNA into complementary DNA (cDNA) before PCR amplification. The necessity for this step confirms the virus as an RNA virus.

Gel Electrophoresis: Following amplification, gel electrophoresis is employed to visualize the nucleic acid strands. This technique allows for the determination of the size and, indirectly, the strandedness of the nucleic acid. Single-stranded nucleic acids often migrate differently through the gel compared to their double-stranded counterparts.

Nucleic Acid Sequencing: Advanced sequencing technologies can provide definitive evidence of the type and nature of the viral nucleic acid. Sequencing can identify the exact nucleotide sequence of the viral genome, confirming whether it is made of DNA or RNA and revealing if it is single-stranded or double-stranded.

Hybridization Techniques: Techniques such as Northern blotting for RNA and Southern blotting for DNA can also be used to determine the type of nucleic acid in the virus. These methods involve the transfer of nucleic acids to a membrane followed by hybridization with a labeled probe specific to DNA or RNA, providing insights into the nature of the viral genome.

Use of Electron Microscopy: In some cases, electron microscopy can offer clues about the viral genome's nature based on the virus's morphology and structure, though this method is less direct and often supplementary to molecular techniques.

Enzymatic Methods: The presence of certain enzymes, such as reverse transcriptase in retroviruses, can also indicate the type of nucleic acid. These enzymes are characteristic of certain RNA viruses and can be detected through specific assays.

In conclusion, the determination of whether a virus is a DNA or RNA virus, and whether it possesses single-stranded or double-stranded nucleic acid, requires a comprehensive approach that combines several laboratory techniques. Each method contributes to a body of evidence that, when taken together, allows for the accurate classification of the viral genome. This scientific process is critical for understanding the virus's biology, pathogenicity, and for developing targeted treatments and interventions.

Sincerely,

[Your Name]

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You

How can I determine the %NH3 in an ammonium salt solution other than by a back titration? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Reader,

Determining the percentage of ammonia (��3NH3) in an ammonium salt solution requires precise analytical techniques. While back titration is a common method, there are alternative approaches that offer accuracy and specificity for quantifying ��3NH3 content. Here, we delve into several methodologies that serve as alternatives to back titration:

Direct Titration with a Strong Acid: Unlike back titration, direct titration involves adding a strong acid, such as hydrochloric acid (HCl), directly to the ammonium salt solution. The endpoint is reached when all the ammonia has reacted with the acid, which can be detected using an appropriate pH indicator or a pH meter. This method provides a straightforward means of calculating the ��3NH3 concentration based on the stoichiometry of the reaction and the volume of acid used.

Ion-Selective Electrode (ISE) Measurement: The use of an ammonium ion-selective electrode provides a rapid and selective measurement of the ��4+NH4+ concentration in solution, which is in equilibrium with ��3NH3. The electrode response, typically in millivolts, is proportional to the logarithm of the ��4+NH4+ ion concentration. This method requires calibration with standards of known ammonium concentration for accurate results.

Spectrophotometric Analysis: Certain chemical reactions result in products with distinct absorbance properties. By reacting the ammonium ion with reagents that produce a colored complex, the concentration of ��3NH3/��4+NH4+ can be determined spectrophotometrically. The intensity of the color, measured at a specific wavelength, correlates with the ammonia concentration in the solution.

Gas Sensing Technologies: Technologies such as chemiresistors, which change their electrical resistance in the presence of specific gases, can be used to detect ��3NH3 gas evolved from the solution. This method involves heating the solution to convert ��4+NH4+ to ��3NH3 gas, which is then quantified using the sensor. The change in resistance provides a measure of the ��3NH3 concentration.

Conductometric Analysis: This technique measures the change in electrical conductivity of the solution as the ammonium ion is converted into ammonia. The conductivity change, resulting from the reaction of the ammonium salt with a strong base to release ��3NH3, can be used to calculate the concentration of ammonia in the solution.

Nessler's Reagent Method: A colorimetric method involving Nessler's reagent, which forms a brownish-yellow complex with ammonia, can be used for the qualitative and semi-quantitative determination of ��3NH3 in solution. The intensity of the color, which can be measured spectrophotometrically, correlates with the ammonia concentration.

Each of these methods offers distinct advantages and may be chosen based on the specific requirements of accuracy, sensitivity, and available laboratory equipment. It is essential to consider the nature of the sample, the concentration range of ��3NH3, and the presence of interfering substances when selecting an appropriate analytical technique.

In conclusion, a variety of analytical methods are available for determining the percentage of ��3NH3 in an ammonium salt solution. The choice of method depends on the specific requirements of the analysis, including accuracy, sensitivity, and the available laboratory resources.

Sincerely,

[Your Name]

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You

给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

It appears that I've already provided a detailed and professional response tailored to your request regarding the determination of %NH3 in an ammonium salt solution. If you have any more questions or need further clarification on a different topic, please feel free to ask!

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You

What is the optimum time to harvest Lentivirus from HEK293T cells after transfection? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

The process of harvesting Lentivirus from HEK293T cells after transfection is a critical step in ensuring the efficiency and viability of the viral particles for subsequent applications. The optimal timing for the collection of Lentivirus post-transfection is contingent upon a myriad of factors including the transfection method, the specific lentiviral construct, and the purpose of the lentivirus. However, based on standard practices and empirical evidence from a broad spectrum of research and protocols, the following guidelines can be recommended.

Typically, the optimal time frame to begin harvesting Lentivirus from HEK293T cells is approximately 48 to 72 hours post-transfection. This window allows sufficient time for the transcription and assembly of viral particles within the host cells and their subsequent release into the culture medium. Here's a more detailed breakdown:

48 Hours Post-Transfection: At this juncture, a significant number of viral particles have been produced and released into the medium. Collecting the virus at this point can yield a substantial amount of viral particles, though waiting slightly longer may increase the yield.

72 Hours Post-Transfection: Harvesting at 72 hours post-transfection is widely regarded as the optimum time for many protocols. The extended period allows for maximal viral production and release, ensuring a higher titer of the lentivirus in the supernatant. Beyond this point, the viability of the HEK293T cells may start to decline, potentially contaminating the viral stock with cellular debris.

Considerations for Extended Harvesting: In some cases, extending the collection period beyond 72 hours, up to 96 hours post-transfection, might be beneficial, especially if the initial viral yield is low or if the specific lentiviral construct has slower kinetics of viral particle production. This extended period should be approached with caution to avoid the degradation of viral particles and to minimize contamination.

For the highest quality of lentiviral preparation, it is recommended to perform a series of collections at different time points within the 48 to 72-hour window. This approach allows for the evaluation of viral titer across different harvesting times, enabling the identification of the optimal collection point for your specific experimental setup.

Additionally, after the initial harvest, it is advisable to filter the collected viral supernatant through a 0.45 µm low protein binding filter to remove cellular debris and to proceed with concentration steps, if necessary, to increase the viral titer.

In conclusion, while the 48 to 72-hour post-transfection window is generally optimal for harvesting Lentivirus from HEK293T cells, specific conditions and experimental requirements may necessitate adjustments. Continuous monitoring and evaluation of viral production, coupled with adherence to best practices in viral handling and safety protocols, are paramount for successful lentiviral vector production.

Best regards,

[Your Name]

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You

Do I need to sequence DNA after every transformation? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Researcher,

The necessity of sequencing DNA after every transformation is contingent upon the objectives of your experiment, the nature of the transformation, and the reliability of the cloning vectors and host systems employed. While not always mandatory, sequencing post-transformation serves as a robust verification step to ensure the accuracy and integrity of the genetic material of interest. Below, we delineate the considerations that should guide your decision on whether to sequence DNA after each transformation event:

Objective of the Experiment: If the goal of your transformation is to introduce a specific mutation, insert, or to construct a novel plasmid, sequencing is indispensable. This verification step confirms the presence and correctness of the intended genetic modification.

Reliability of Cloning Method: Certain cloning techniques, such as those employing high-fidelity assembly methods or site-specific recombination systems, have lower error rates. However, even with these methods, sequencing is recommended to verify the assembly, especially for complex constructs or when mutations are introduced.

Previous Validation of Constructs: If you are transforming a well-characterized and previously validated construct under the same conditions, you might not need to sequence after every transformation. This is particularly relevant for routine procedures where the vector and the host strain have demonstrated consistent performance.

Screening Strategies: When a high-throughput screening strategy is employed, it may not be practical to sequence every clone. Instead, initial screening might involve functional assays or restriction analysis to narrow down candidates, followed by sequencing of selected clones to confirm the correct sequence.

Resource Considerations: Sequencing can be resource-intensive in terms of time and cost. The decision to sequence might also be influenced by the availability of resources and the critical nature of the experiment. For critical or novel experiments, sequencing is a worthwhile investment to ensure the validity of the results.

Regulatory and Publication Requirements: For experiments that will contribute to regulatory submissions or publications, sequencing to confirm the identity and integrity of the transformed DNA is often required to meet the rigorous standards of proof for scientific reporting.

In conclusion, while not every transformation necessitates subsequent DNA sequencing, it is a critical step for confirming the success of genetic modifications and ensuring the reliability of your experimental results. The decision to sequence should be based on a balanced consideration of experimental goals, the cloning method's reliability, resource availability, and the downstream applications of the transformed material.

Yours sincerely,

[Your Name]

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You

Coronavirus and its impact on peoples life all around the world! 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Reader,

The coronavirus pandemic, caused by the SARS-CoV-2 virus, has had a profound and unprecedented impact on global society, affecting every aspect of human life. Emerging in late 2019 and declared a pandemic by the World Health Organization (WHO) in March 2020, its repercussions have been felt worldwide, leading to significant health, economic, social, and psychological challenges. Here, we explore the multifaceted impact of the coronavirus on people's lives around the globe.

Health Impact: The most immediate and visible effect of the coronavirus has been on global health. Millions of individuals have been infected, leading to a significant number of fatalities. The healthcare systems in many countries have been overwhelmed, with hospitals operating beyond capacity and facing shortages of essential medical supplies and personnel. The pandemic has also highlighted and exacerbated existing inequalities in healthcare access and outcomes.

Economic Consequences: Economically, the pandemic has triggered a global recession, affecting economies at both the macro and micro levels. Businesses, particularly small and medium-sized enterprises, have faced closures and financial hardships, leading to unprecedented levels of unemployment and economic instability. The disruption of global supply chains and trade has further compounded these economic challenges.

Social and Psychological Effects: On a social level, measures to control the virus's spread, such as lockdowns, social distancing, and travel restrictions, have transformed everyday life, affecting relationships, education, and work. The pandemic has led to increased social isolation and has taken a toll on mental health, contributing to higher rates of anxiety, depression, and other psychological issues.

Educational Disruption: The education sector has experienced significant disruptions, with schools and universities worldwide shifting to online learning. This sudden change has highlighted and widened the digital divide, affecting students' learning outcomes, particularly those from disadvantaged backgrounds.

Work and Employment Shifts: The pandemic has reshaped the work landscape, accelerating the shift towards remote work and digital transformation. While this has offered flexibility and safety for some, it has also led to job losses in sectors that cannot easily transition to remote work, further exacerbating employment inequalities.

Global Inequality: The pandemic has magnified existing inequalities within and between countries. Access to healthcare, vaccines, and economic support has varied greatly, with lower-income countries and disadvantaged populations facing greater challenges in coping with the pandemic's impacts.

Innovation and Adaptation: Despite the challenges, the pandemic has also spurred innovation and adaptation across various sectors. The rapid development of vaccines is a testament to scientific progress, while businesses and educational institutions have found new ways to operate and engage with their audiences.

In conclusion, the coronavirus pandemic has had a profound impact on the fabric of global society, affecting health, economy, education, and daily life. While it has presented significant challenges, it has also highlighted the importance of resilience, adaptability, and global cooperation in addressing complex global issues. The long-term effects of the pandemic will likely continue to shape the world in the years to come.

Yours sincerely,

[Your Name]

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You

How well does Cas9 gene targeting work in bacteria or archaea? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

The application of CRISPR-Cas9 gene editing technology in bacteria and archaea represents a significant advancement in microbial genetics and biotechnology. Originating as an adaptive immune system in these microorganisms, the Cas9 endonuclease has been repurposed for targeted genetic modifications with remarkable efficiency. Here, we provide an overview of the efficacy of Cas9 gene targeting in bacteria and archaea, underpinned by key considerations, successes, and challenges.

Mechanism and Efficacy: Cas9-mediated gene editing operates by inducing double-strand breaks (DSBs) at specific genomic locations, guided by a complementary RNA sequence. The cell's native DNA repair mechanisms then act on these breaks, allowing for targeted gene disruption, insertion, or modification. In bacteria and archaea, the system's native origin ensures a high level of compatibility and efficacy. However, the efficiency of Cas9 gene targeting can vary significantly depending on several factors:

Species-Specific Factors: The success of Cas9 gene editing is highly dependent on the host species' cellular machinery. Factors such as the efficiency of RNA processing, the presence and activity of endogenous nucleases, and the capacity for homologous recombination can all influence outcomes.

Delivery Method: Efficient delivery of the Cas9 protein and its guide RNA (gRNA) into microbial cells is crucial. Methods such as transformation, electroporation, or viral vectors have been employed, with varying degrees of success across different species.

Target Sequence and gRNA Design: The specificity and efficacy of Cas9 targeting are heavily influenced by the target sequence and the design of the guide RNA. Optimal gRNA design minimizes off-target effects while maximizing on-target activity.

Repair Mechanism Utilization: The outcome of Cas9-induced DSBs depends on the cell's DNA repair pathways. In bacteria and archaea, the predominance of non-homologous end joining (NHEJ) or homologous recombination (HR) mechanisms can affect the efficiency of precise gene editing.

Applications and Success Stories: Cas9 gene targeting has been successfully applied in various bacteria and archaea for both fundamental research and biotechnological applications. These include gene function studies, metabolic engineering, the development of novel antibiotics, and the construction of microbial cell factories for the production of valuable compounds. In particular, the system has shown great promise in genetically intractable or less-studied species, opening new avenues for research and application.

Challenges and Future Directions: Despite its successes, Cas9 gene targeting in bacteria and archaea faces challenges. These include the potential for off-target effects, variability in editing efficiency across different species, and the need for optimized delivery methods. Ongoing research is focused on addressing these challenges through the development of improved Cas9 variants with higher specificity, the exploration of alternative CRISPR systems, and the creation of more efficient delivery mechanisms.

In conclusion, Cas9 gene targeting has proven to be a powerful tool for genetic manipulation in bacteria and archaea, with wide-ranging applications in research and biotechnology. While challenges remain, the continued refinement of this technology holds great promise for advancing our understanding and utilization of microbial systems.

Yours sincerely,

[Your Name]

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You

PGK promoter acting as a bidirectional promoter? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Researcher,

Your inquiry regarding the functionality of the phosphoglycerate kinase (PGK) promoter as a bidirectional promoter is quite intriguing. The PGK promoter is widely recognized for its robustness and constitutive expression in various host cells, primarily used in mammalian expression systems. Typically, promoters are considered to be unidirectional, driving the expression of genes placed downstream in a specific orientation. However, the concept of bidirectional promoters, which can initiate transcription in both directions, opens up fascinating avenues for gene expression studies and vector design.

The PGK promoter, derived from the gene encoding phosphoglycerate kinase, is predominantly characterized as a strong, constitutive promoter facilitating high levels of gene expression. Its use is extensive in the field of genetic engineering and biotechnology for the expression of recombinant proteins and for gene therapy applications.

To address the question of its bidirectionality, it's essential to understand that a bidirectional promoter contains elements that can initiate transcription on both strands of DNA, leading to the production of two different RNA molecules from the two opposite strands. This characteristic is not commonly attributed to the classical understanding of the PGK promoter's functionality. Bidirectional promoters are typically found in the genome where two genes are located head-to-head, with a relatively short intergenic region between them. These promoters have sequences that are recognized by the transcription machinery in both directions.

While the conventional use of the PGK promoter does not explicitly include bidirectional expression, the concept of engineering promoters or utilizing naturally occurring bidirectional promoters has gained traction. In contexts where the PGK promoter has been manipulated or combined with other elements to drive bidirectional expression, it would represent a novel application rather than its inherent characteristic.

Furthermore, the exploitation of bidirectional promoters, whether naturally occurring or synthetic, offers a compact and efficient means to co-express genes, which is particularly advantageous in vector design for gene therapy, functional genomics, and synthetic biology.

In summary, while the PGK promoter is primarily known and used for its strong, unidirectional expression capabilities in eukaryotic systems, the concept of bidirectional promoters, including potentially modified versions of the PGK promoter, represents an area of ongoing research and development. The exploration into bidirectional promoters, including engineering existing promoters like PGK for such purposes, holds significant potential for advancements in gene expression technologies and multi-gene manipulation strategies.

Yours sincerely,

[Your Name]

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You

How do I estimate the Kd of a high affinity protein using only instrinsic tryptophan fluorescence spectroscopy? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

Estimating the dissociation constant (Kd) of a high-affinity protein-ligand interaction using intrinsic tryptophan fluorescence spectroscopy is a sophisticated approach that leverages the sensitivity of tryptophan residues to their microenvironment. This method is particularly useful when tryptophan residues are located near the binding site, as their fluorescence properties will change upon ligand binding, providing a quantitative measure of the interaction. Below is a structured methodology to accurately determine the Kd of a high-affinity protein using this technique:

Preparation of Protein and Ligand Solutions: Begin by preparing solutions of your protein of interest and the ligand in a suitable buffer. It is crucial to ensure that the protein is purified and the tryptophan residues are not quenched by the buffer components.

Baseline Fluorescence Measurement: Measure the fluorescence emission spectrum of the protein alone to establish a baseline. Tryptophan typically exhibits maximum fluorescence emission between 330 and 350 nm when excited at 280 nm. Ensure the instrument settings are optimized for sensitivity and reproducibility.

Incremental Addition of Ligand: Add small, incremental concentrations of the ligand to the protein solution, allowing the system to equilibrate after each addition. It is essential to keep the protein concentration constant throughout the experiment to ensure that changes in fluorescence are due to ligand binding and not alterations in protein concentration.

Recording Fluorescence Changes: After each addition of ligand, record the fluorescence emission spectrum. Pay particular attention to shifts in the maximum emission wavelength and changes in fluorescence intensity, as these indicate binding events.

Data Analysis: Analyze the fluorescence intensity changes as a function of ligand concentration. For high-affinity interactions, you will observe a saturation curve where fluorescence changes plateau at high ligand concentrations, indicating that most binding sites are occupied.

Kd Estimation: To estimate the Kd, fit your data to an appropriate binding model, such as the Langmuir binding isotherm. This model relates the fluorescence intensity change (ΔF) to the ligand concentration [L], the maximum fluorescence change (ΔFmax), and the Kd:Δ�=Δ�max⁡[�]��+[�]ΔF=ΔFmaxKd+[L][L]Nonlinear regression software can be used to fit the curve and extract the Kd value. Ensure the fitting process accounts for the high-affinity nature of the interaction, which may require precise measurement of low ligand concentrations.

Validation and Replication: Validate the estimated Kd by replicating the experiment under identical conditions to ensure the accuracy and reliability of your measurements. Consideration of potential sources of error, such as non-specific binding or changes in protein conformation, is crucial for accurate interpretation.

Intrinsic tryptophan fluorescence spectroscopy provides a powerful and sensitive method for investigating protein-ligand interactions, especially for high-affinity complexes. The precision in preparation, measurement, and analysis is paramount to accurately determining the Kd value, offering insights into the binding dynamics and molecular mechanisms governing the interaction.

Yours sincerely,

[Your Name]

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You

Intravenous injection of herbal extracts in rodents acceptable or not? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

The administration of herbal extracts via intravenous injection in rodents is a subject that necessitates careful consideration, particularly with regard to the safety, ethical implications, and scientific validity of such procedures. Intravenous injection is a direct and efficient method to deliver substances into the bloodstream, allowing for immediate systemic distribution. However, when it comes to herbal extracts, several key factors must be meticulously evaluated:

Purity and Standardization of Herbal Extracts: Herbal extracts are complex mixtures containing multiple bioactive compounds, whose concentrations can vary significantly depending on the source, extraction method, and storage conditions. Prior to administration, it is essential to ensure the extract is of high purity and is standardized to contain consistent levels of the active constituents.

Safety and Toxicity: The safety profile of the herbal extract must be thoroughly assessed through in vitro and smaller scale in vivo studies before proceeding to intravenous administration in rodents. Many compounds safe for oral consumption may have different toxicity profiles when bypassing the digestive system's metabolic processes.

Solubility and Vehicle: The solubility of the herbal extract in the vehicle used for injection is critical. Insoluble particles can lead to embolism or acute inflammatory responses. The choice of vehicle should also be compatible with intravenous administration, not causing hemolysis or adverse reactions.

Dose Determination: Establishing the appropriate dosage is crucial to avoid toxic effects. Dose-finding studies should be conducted to determine the maximum tolerated dose and the therapeutic dose range.

Ethical Considerations: All experimental procedures involving animals should be designed with the utmost attention to ethical considerations. This includes minimizing pain and distress, using the minimum number of animals necessary to achieve scientific objectives, and ensuring that the study is scientifically justified and designed to produce valuable knowledge.

Regulatory Compliance: Compliance with institutional, national, and international guidelines and regulations governing animal research is mandatory. This often involves obtaining approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent body, which evaluates the scientific and ethical aspects of the proposed research.

Monitoring and Welfare: Continuous monitoring of the animals for adverse effects following the injection is imperative to promptly address any health issues. Protocols should be in place for the management of unexpected reactions to ensure animal welfare.

In conclusion, while the intravenous injection of herbal extracts in rodents can be scientifically valuable, it requires rigorous preparation, safety assessment, and ethical considerations. The acceptability of such a method depends on the ability to meet these stringent criteria, ensuring that the research is conducted responsibly, safely, and with clear scientific merit.

Yours sincerely,

[Your Name]

📷

You

Which of the following structures contains genes for enzymes and antibiotic resistance? Plasmid Pilus Capsule Plasma Membrane ? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

Among the structures listed — plasmid, pilus, capsule, and plasma membrane — the entity that contains genes for enzymes and antibiotic resistance is the plasmid. Plasmids are small, circular, double-stranded DNA molecules that are distinct from a bacterial cell's chromosomal DNA. They are capable of autonomous replication and often carry genes that may confer advantageous traits to bacteria, such as antibiotic resistance and the production of enzymes that degrade toxic compounds.

Plasmids play a pivotal role in horizontal gene transfer among bacterial populations, significantly contributing to the spread of antibiotic resistance. This characteristic makes them subjects of intense study in the context of infectious disease research and microbial ecology.
Pili (plural of pilus), on the other hand, are filamentous structures on the surface of bacterial cells that facilitate attachment to surfaces and other cells. While they are crucial for processes such as bacterial conjugation, during which plasmids can be transferred between cells, they do not themselves contain genes.
Capsules are gelatinous layers that encase some bacterial cells, providing protection against desiccation and phagocytosis. Although capsules play a role in bacterial virulence, they do not contain genetic material.
The plasma membrane is a phospholipid bilayer that encloses the cell, controlling the passage of substances in and out of the cell. While essential for numerous cellular functions, it does not house genes.

In conclusion, plasmids are the structures among those listed that contain genes for enzymes and antibiotic resistance, underscoring their significance in bacterial adaptation and survival, especially in environments with selective pressures such as antibiotics.

Yours sincerely,

[Your Name]

📷

You

Can anyone suggest me appropriate cell lysis buffer for extract viral DNA from virus infected cells? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

When it comes to extracting viral DNA from virus-infected cells, selecting the appropriate cell lysis buffer is crucial to ensure efficient lysis of cells, preservation of viral DNA, and minimization of contaminating DNA from the host. The choice of lysis buffer can vary depending on the type of virus and the nature of the infected cells. However, a commonly used and effective lysis buffer for this purpose is a Nonidet P-40 (NP-40) buffer or a buffer containing Tris-HCl, EDTA, and SDS. Here is a detailed breakdown of these recommendations:

NP-40 Buffer: This is a non-ionic detergent-based buffer that gently disrupts cellular membranes, allowing for the release of cytoplasmic contents without extensively shearing DNA. An example formulation might include:150 mM NaCl 1.0% NP-40 (Nonidet P-40 substitute) 50 mM Tris-HCl, pH 8.0

Tris-EDTA (TE) Buffer with SDS: For a more robust cell lysis, especially when dealing with tougher or more resilient cells, a buffer containing SDS can be used. SDS is an ionic detergent that denatures proteins and disrupts cellular and nuclear membranes more effectively. A typical buffer might contain:10 mM Tris-HCl, pH 8.0 1 mM EDTA, pH 8.0 0.5% SDS

It's important to note that while NP-40 provides milder lysis conditions, which can be advantageous for preserving viral integrity, SDS-based buffers can lead to more complete lysis but may also result in higher shear forces on the DNA.

Additional Considerations:

Proteinase K Treatment: Including Proteinase K in the lysis buffer can aid in degrading proteins and ensuring that the viral DNA is free from protein contamination. Proteinase K is particularly useful when using SDS in the lysis buffer, as it works well in the presence of detergents.
RNase Treatment: To remove RNA contamination, RNase can be added to the lysis buffer. This step is critical for ensuring that the extracted nucleic acid is predominantly viral DNA.
Phenol-Chloroform Extraction and Ethanol Precipitation: Following lysis, phenol-chloroform extraction and ethanol precipitation are commonly used to purify the viral DNA, removing proteins and other contaminants.

In conclusion, the choice of cell lysis buffer should be tailored to the specific requirements of your viral DNA extraction, considering the nature of the virus and the host cells. Both NP-40 based buffers and SDS-containing buffers offer effective solutions, with the decision guided by the need for gentle lysis versus more robust extraction conditions. The inclusion of Proteinase K and RNase treatment further enhances the purity of the viral DNA, ensuring high-quality samples for subsequent analyses.

Yours sincerely,

[Your Name]

📷

You

Different types of COVID-19 related virus genome sequence from different regions? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has seen the emergence of multiple variants of the virus, each with distinct genomic sequences. These variants have been identified through extensive genomic surveillance and sequencing efforts across different regions worldwide. The variations in the virus genome sequence are a result of mutations, which can lead to changes in the virus's properties, such as transmissibility, disease severity, and vaccine efficacy. Here, we outline the major types of SARS-CoV-2 variants that have been documented:

Alpha Variant (B.1.1.7): First identified in the United Kingdom, the Alpha variant has mutations in the spike protein, which may increase the virus's ability to bind to host cells, making it more transmissible.

Beta Variant (B.1.351): Emerging in South Africa, this variant carries mutations that not only enhance transmissibility but also potentially reduce the efficacy of antibodies generated through previous infection or vaccination.

Gamma Variant (P.1): Initially detected in Brazil, the Gamma variant shares some mutations with the Beta variant, suggesting possible impacts on antibody neutralization and increased transmissibility.

Delta Variant (B.1.617.2): First found in India, the Delta variant is associated with higher transmissibility and has been linked to increased hospitalization rates. It contains mutations in the spike protein that facilitate cell entry and evade some antibody responses.

Omicron Variant (B.1.1.529): Identified in South Africa, the Omicron variant is characterized by a large number of mutations, especially in the spike protein. These mutations are associated with significant changes in the virus's behavior, including potentially higher transmissibility and impacts on the effectiveness of vaccines.

Each of these variants has been categorized based on the genetic changes they possess, which have implications for public health responses, vaccine development, and treatment strategies. The World Health Organization (WHO) and other health authorities continuously monitor these variants, classifying them as "Variants of Concern" (VOCs) or "Variants of Interest" (VOIs) based on their impact on transmission, disease severity, and the effectiveness of public health and social measures.

The diversity in the COVID-19 virus genome sequences across different regions underscores the importance of ongoing genomic surveillance and research to understand the evolving nature of the virus. This information is crucial for adapting public health strategies, updating vaccines, and developing therapeutics to combat the COVID-19 pandemic effectively.

Yours sincerely,

[Your Name]

📷

You

Does it matter if we propagate influenza A virus in DMEM instead of MEM? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

The choice of cell culture medium, such as Dulbecco's Modified Eagle Medium (DMEM) versus Minimum Essential Medium (MEM), can indeed influence the propagation of influenza A virus in vitro. Both DMEM and MEM are widely used for cell culture applications, including the cultivation of viruses, but they differ in their formulation, including the concentration of amino acids, vitamins, and other nutrients. These differences can affect the efficiency of virus replication, the health and maintenance of the host cells, and ultimately, the yield of the virus. Here are several considerations to keep in mind:

Nutrient Composition: DMEM generally contains a higher concentration of amino acids and vitamins compared to MEM. This enriched nutrient composition can support the more robust growth of certain cell lines, which might be advantageous for virus propagation.

Cell Line Specific Requirements: The optimal growth conditions for the host cell line used for virus propagation, such as MDCK (Madin-Darby Canine Kidney) cells commonly used for influenza virus, may differ. Some cell lines might perform better in DMEM, while others might prefer MEM. The health and metabolic state of the host cells are critical for efficient viral replication.

Virus Strain Specificity: Different strains of influenza A virus may have varying requirements for optimal replication. The choice of medium should ideally be based on empirical evidence or literature supporting its use for the specific strain of influenza A virus being studied.

Experimental Consistency: For comparative studies or longitudinal research, maintaining consistency in the culture medium is essential to ensure that observed differences in viral behavior or response to treatments are not attributed to variations in the culture conditions.

Supplementation Needs: Both DMEM and MEM can be supplemented with additional components, such as serum, antibiotics, or specific growth factors, to enhance cell growth and virus production. The requirement for and type of supplementation may influence the choice of base medium.

In summary, while both DMEM and MEM can be used for propagating influenza A virus, the choice between them should be informed by the specific requirements of the host cell line, the particular strain of the virus, and the objectives of the experiment. It is advisable to consult existing literature or perform preliminary experiments to determine the most suitable medium for your specific needs. Adjustments to the culture conditions, including the medium formulation, should be carefully documented and considered in the analysis and interpretation of the results.

Yours sincerely,

[Your Name]

📷

You

Is there a way to check whether Ligation has worked properly before doing transformation? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

Ensuring the success of a ligation reaction before proceeding to the transformation step is a prudent practice in molecular biology. It not only saves time and resources but also increases the likelihood of a successful experiment. There are several methods to verify the efficiency of a ligation reaction prior to transformation. Here are a few strategies:

Agarose Gel Electrophoresis: The most straightforward method to check ligation efficiency is by running an aliquot of your ligation mixture on an agarose gel alongside control samples. By comparing the migration pattern of your ligated product to that of unligated vector and insert, you can often infer whether ligation has occurred. Successful ligation typically results in a shift in molecular weight that is visible on the gel.

Control Ligation Reactions: Including control reactions in your experiment can help determine the success of the ligation. A positive control (using a vector and insert known to ligate successfully) and a negative control (vector without ligase) can provide benchmarks for comparison. Absence of ligation in the negative control and successful ligation in the positive control can indicate that the ligation conditions are effective.

Restriction Enzyme Digest: For vectors and inserts cut with different restriction enzymes, performing a post-ligation restriction digest can help verify the presence of the insert within the vector. The pattern of bands observed after digesting the ligation product can indicate successful ligation if it matches the expected sizes for a correctly ligated construct.

PCR Amplification: Performing PCR on the ligation mixture using primers flanking the cloning site can also indicate successful ligation. The presence of a PCR product of the expected size suggests that the insert has been successfully ligated into the vector.

Quantitative Real-Time PCR (qPCR): Although more sophisticated and less commonly used for this purpose, qPCR can be employed to quantify the amount of ligated product, providing a more precise assessment of ligation efficiency.

Ligation-Independent Cloning (LIC) Techniques: As a more indirect approach, some researchers opt for ligation-independent cloning methods that do not require ligase, thus bypassing the traditional ligation step. While not a method to check ligation per se, it's an alternative strategy to consider if consistent issues with ligation efficiency are encountered.

It is important to note that while these methods can provide indications of ligation success, the ultimate test of ligation efficiency is often observed in the transformation efficiency and the subsequent identification of correct clones. Therefore, these pre-transformation checks should be viewed as preliminary, albeit valuable, steps in the cloning workflow.

In conclusion, employing one or more of these methods to check the success of your ligation reaction before proceeding to transformation can significantly enhance the efficiency of your cloning experiments. Careful planning and execution of these strategies will contribute to more reliable outcomes and resource savings.

Yours sincerely,

[Your Name]

📷

You

What is the best method for transfecting HEK293 with multiple plasmids in order to generate chimeric HIV virions? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文

ChatGPT

Dear Colleague,

Transfecting HEK293 cells with multiple plasmids to generate chimeric HIV virions is a critical step in studying viral assembly, entry, replication, and host-virus interactions. The choice of transfection method can significantly impact the efficiency of plasmid delivery and subsequent viral production. Based on current best practices in the field, the calcium phosphate precipitation method, lipofection, and electroporation are among the most effective methods for transfecting HEK293 cells with multiple plasmids. Here, we delve into the advantages and considerations of each approach:

Calcium Phosphate Precipitation: This method is widely used for transfecting HEK293 cells due to its cost-effectiveness and efficiency in delivering multiple plasmids into cells. The calcium phosphate method involves mixing plasmid DNA with calcium chloride and then adding it to a phosphate buffer to form a precipitate that is taken up by the cells. Although highly effective, this method requires optimization of DNA concentration and precipitate formation time to maximize transfection efficiency while minimizing cytotoxicity.

Lipofection: Utilizing liposomal transfection reagents (e.g., Lipofectamine) provides another highly efficient method for transfecting multiple plasmids into HEK293 cells. Lipofection involves encapsulating the plasmid DNA in liposomes, which then fuse with the cell membrane, facilitating DNA entry into the cytoplasm. This method is known for its high transfection efficiency and ease of use. However, it is more costly than calcium phosphate precipitation and may require optimization to identify the most effective liposome-to-DNA ratio.

Electroporation: Electroporation uses an electrical field to transiently permeabilize the cell membrane, allowing plasmid DNA to enter the cell. This method is suitable for transfecting large quantities of plasmid DNA and can be highly efficient, particularly for difficult-to-transfect cells. The main considerations for electroporation include optimizing the electrical parameters and the amount of DNA to achieve high transfection efficiency without compromising cell viability.

Key Considerations for Generating Chimeric HIV Virions:

Plasmid Ratio: When transfecting multiple plasmids, it is crucial to optimize the ratio of envelope, gag-pol, and transfer vector plasmids to ensure efficient packaging and production of chimeric virions.
Transfection Efficiency: Regardless of the method chosen, achieving high transfection efficiency is paramount. This often requires careful optimization of the transfection conditions specific to the HEK293 cell line and the plasmids being used.
Cell Health: Maintaining the health and viability of HEK293 cells is essential for successful transfection and viral production. This includes optimal cell density at the time of transfection and appropriate post-transfection care.
Safety Considerations: Working with HIV-derived components necessitates strict adherence to biosafety protocols to prevent accidental exposure and ensure safe handling of potentially infectious materials.

In conclusion, while each of the methods mentioned has its advantages and limitations, the choice should be guided by the specific requirements of your experiment, including the number and size of plasmids, available resources, and desired efficiency. It may also be beneficial to perform preliminary experiments to compare the efficiency of different transfection methods under your specific experimental conditions.

Yours sincerely,

With this protocol list, we might find more ways to solve this problem.

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