Transfection - Science method

Why don't you use commercial products? lipofectamin, Mirus, jetpei.... there is plenty of them try the one that is best for your cells...

Hi Lia,

I hope all is well. Since this is an old post, I am not sure if you still work on the Expi cells and PEI transfection. I am currently doing a large-scale Expi cell suspension culture and will use PEI MAX for the transfection. I have my seed culture growing with 1xPen-Strep (1x defined as 100 units/mL), and am considering diluting the culture into antibiotic-free medium for spliting purposes, which will reduce the Pen-Strep concentration in the culture. Do you think 0.02x Pen-strep concentration in the final culture will interfere with the transfection?

Thanks for the advice. The knock out mouse is not available but I will try the lentivirus .

Tripti

What bacterial species are you working with? There's many GFP/RFP plasmids out there, but which ones you use depend on what species you're talking about. Plasmid replication, promoter recognition and codon usage is generally restricted by taxonomy.

Generally, my favorite place to obtain plasmids is AddGene.org

CRISPR-Cas9-mediated gene deletion is a powerful tool for genome editing, allowing precise removal of specific genomic regions. The best approach for CRISPR-Cas9-mediated gene deletion may vary depending on the specific experimental requirements, cell type, and desired outcome. However, careful design of gRNAs, efficient delivery of CRISPR components, thorough screening and validation, and rigorous off-target analysis are key considerations for successful gene deletion experiments. Additionally, optimization and iterative refinement of experimental procedures can help improve the efficiency and reliability of CRISPR-Cas9 editing.

Certainly, I can guide you on how to evaluate and analyze the cytopathic effect (CPE) caused by adenoviruses in cell cultures, which is crucial for identifying virus infection and determining its intensity. Adenovirus CPE involves characteristic changes in host cells induced by viral invasion, which can be used effectively to assess the progress and severity of infection. Here’s a detailed approach to study adenovirus CPE:

This structured approach allows for a thorough investigation of adenovirus cytopathic effects, providing valuable insights into the virus-cell interactions and the efficacy of potential antiviral treatments. Regular and meticulous monitoring coupled with detailed documentation are key for a successful analysis of adenovirus CPE.

Reviewing the protocols listed here may offer further guidance in addressing this issue

Regarding assessing the expression of specific mutants, I typically refer to primers used in several papers that describe the design of specific primers for detecting mutant expression. In addition, you can consider to perform RT-qPCR using two sets of primers: one targeting the wildtype sequence, and the other targeting the mutant sequence.

Using a different grade of cells for your positive control could potentially introduce variability or inconsistency into your experiment. It's generally recommended to use the same grade or quality of cells for both experimental and control groups to ensure reliable and accurate results. However, if you have valid reasons for using a different grade of cells for your positive control, it's essential to thoroughly validate and justify this decision to ensure the integrity of your experimental findings.

Sabine Strehl thank you for answering! Under G418 selection pressure, the resistant cells did not die but not exert their typical morphology even after 10 days. The non resistant cells die 99% but not completely, as I reduce the antibiotic level the non resistant cells overgrowth than the resistant cells that express EGFP and slowly my resistant cells reduce and die off and non resistant cells overgrowth. I use medium with G418 for 7 days then prepare a fresh one, but on 7th day of the medium, the antibiotic seems wear off the potency after a few water bath thaw.

I will repeat the transfection and selection process. Thank you for your suggestion.

Fluorescence Recovery After Photobleaching (FRAP) is a technique used in cell biology and microscopy to study the dynamics of molecules within living cells. Here's how it works:

FRAP is a powerful tool for understanding the spatiotemporal dynamics of molecules in living cells, providing insights into their behavior and function in complex biological systems.

honestly whatever works, works. Try out electroporation and see what happens.

I wanted to extend my heartfelt thanks for taking the time to respond to my question. Your insights were incredibly valuable and have provided me with a deeper understanding of the topic. Your willingness to share your expertise is truly appreciated.

if "there's no promotor before my target gene"... then you have the answer

It is difficult to have an single optimal way for every cell lines, each cells have their own strength driving multiple signaling pathway, upper limit of the plasmid copy received and interaction between GOI and its own proteins, hence affecting gene expression facility. We had tried at least four different transfection method with more than ten different reagents just to make a cell population to express 0.1% of GOI, while in 293T the expression was almost hitting the upper limit of detection in flow cytometry. Therefore, to generate an optimized protocol for each cell lines or primary cells are considered easier then to generate a single protocol for universal use. The cloning process or to achieve a single double KI/KO clone by CRISPR is very time consuming and troublesome, if you want to go through CRISPR, try look at the newest technique such as base/prime editing by Dr. David Liu. In the other hand, lentivirus is not, in our lab, lentivirus is an useful tool for large scale cell line production, a single production process may grant us enough amount of lentiviral particle for more then 100 times of usage. However, if a gene is already being difficult to express in such cell lines, I would rather look into an optimization of DNA construct, a small twist such as codon optimization or driving promoter sometimes grant you more then 50-fold of difference, that will be far practical then transfection process.

Best

Asma El-Howati Yes, that is true. But why bother with unmixing GFP from FITC, if you can simply run the assay using AnnV-APC for instance. The cleaner the setup the better :)

Cheers.

Dear [Recipient],

I hope this message finds you well. Your inquiry regarding the use of liposomes for the delivery of Cas9/gRNA ribonucleoprotein (RNP) complexes and double-stranded oligodeoxynucleotides (dsODNs) is both timely and relevant in the context of genome editing technologies. This delivery strategy is increasingly recognized for its potential to facilitate efficient, targeted genome editing by reducing off-target effects and enhancing delivery efficiency. Below, I provide detailed advice and considerations for employing liposomes in this cutting-edge application.

Understanding the Delivery System:

Liposomes are versatile, lipid-based vesicles capable of encapsulating nucleic acids and proteins, facilitating their delivery into cells. Their biocompatibility, low immunogenicity, and ability to merge with cell membranes make them an attractive delivery system for Cas9/gRNA RNP and dsODNs.

Preparing Cas9/gRNA RNP and dsODNs:

Liposome Preparation and Complex Loading:

Cell Targeting and Uptake:

Monitoring Editing Efficiency and Off-target Effects:

Conclusion:

Liposome-mediated delivery of Cas9/gRNA RNP and dsODNs represents a promising avenue for enhancing the specificity and efficiency of genome editing applications. Success in this approach requires careful consideration of liposome composition, optimization of encapsulation processes, and thorough evaluation of delivery outcomes in target cells. Continued innovation and optimization in this area are expected to further refine these techniques, broadening their applicability and effectiveness in both research and therapeutic contexts.

Should you require further assistance or wish to explore additional aspects of this delivery method, please do not hesitate to reach out. I am here to support your research endeavors.

Best regards,

Take a look at this protocol list; it could assist in understanding and solving the problem.

Hello Ananya Chatterjee

Transfecting macrophages is especially difficult since macrophages are professional phagocytes that are very sensitive to foreign materials. Introduction of foreign DNA activates the Toll-like receptor 9 (TLR9) pathway leading to the production of cytokines and nitric oxide. These activated macrophages may then be less responsive to treatment that one may intend to examine.

You may need to optimize the protocol for various parameters such as timing of transfection, type of transfection reagents, amounts of transfection reagents and of plasmid DNA, as well as ratio of transfection reagent to plasmid DNA.

One critical step for RAW264.7 which you need to consider is the health of the cells. Overgrown cultures are not suitable for transfection as their physiology changes, and continuous culturing of RAW264.7 cells for a long period of time can also change the phenotype and function of the cells. Freshly thawed cells that have a low passage number are recommended for transfection.

Another important consideration is the choice of transfection reagents. Lipid-based transfection reagents are typically used in research due to its ease of use and commercial availability. The cells should be incubated with the transfection reagents for a few hours instead of overnight. Longer incubation time with the transfection reagent will increase transfection efficiency, but it can also be harmful to the cells either causing cell death or cell activation, both of which can interfere with the experimental design. Also, the time between transfection and experimental treatment (the rest time) is crucial.

You may want to refer to the paper attached below. It will be helpful!

Good Luck!

Your gene may be stressing your cells. This may be because the gene is so overly expressed it's consuming too much of the cells' resources, leading to stress and slow growth. The gene itself may be toxic or problematic in some way too. Not knowing the cell line or the gene, it's hard to say what may be happening.

May consider using Permai fluorescence dye.

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and deeply engaged in your research endeavors, particularly those involving the use of Chinese Hamster Ovary (CHO) cells for recombinant protein production. Your inquiry regarding the application of dihydrofolate reductase (DHFR) - methotrexate (MTX) selection system in CHO cells lacking endogenous DHFR activity (DHFR-negative cells) is of significant interest for enhancing gene amplification and expression. Below, I provide a detailed overview of employing this system for the selection and amplification of transfected genes in DHFR-negative CHO cells.

Overview of DHFR-MTX Selection in DHFR-Negative CHO Cells

The DHFR-MTX selection system is a powerful tool for the selection and subsequent amplification of transfected genes in mammalian cell lines, including DHFR-negative CHO cells. This system exploits the enzyme dihydrofolate reductase (DHFR), which is crucial for the synthesis of thymidine and purine nucleotides, thereby enabling DNA synthesis and cell replication.

Key Steps and Considerations

Additional Tips

Conclusion

The DHFR-MTX selection system offers a robust strategy for the selection and gene amplification of transfected DHFR-negative CHO cells, facilitating high-level expression of recombinant proteins. By carefully designing the selection strategy and rigorously screening for high-producing clones, this system can significantly enhance the yield and stability of protein production in CHO cells.

Should you require further assistance or wish to discuss additional strategies for optimizing recombinant protein expression in mammalian cells, please do not hesitate to reach out. I am here to support your scientific journey and contribute to the success of your research projects.

Warm regards.

This protocol list might provide further insights to address this issue.

Hey,

I prepared the buffer.

When I transduce, my neurons are in neurobasal phenolred negative, I cannot check colour change.

But I tried only buffer titration from 1 microliter to 50 microliter. It made no cell death.

May consider using Permai fluorescence dye.

Sure. The settings working the best for me were 1400 voltage, 20 pulse width and pulse number 2. The other one that was working quite fine was 990/40/1 (better transfection but higher mortality) and 1200/40/1 (worse transfection rate but higher viability). Hope you find it helpful!

Never used these two before. But I used Lipofectamine 2000 to deliver the sgRNAs to the Cas9-expressing cells back in 2017, and it worked.

DG44 cells are a type of Chinese hamster ovary (CHO) cell line commonly used in biopharmaceutical research and production, particularly for the production of recombinant proteins and monoclonal antibodies. Several factors can affect the viability of DG44 cells, including:

By carefully optimizing these factors and maintaining appropriate culture conditions, you can enhance the viability and robustness of DG44 cell cultures for various applications in biopharmaceutical research and production.

DNA extraction from cell culture medium can be challenging due to the presence of various contaminants such as proteins, salts, and other cellular debris. However, several methods can be used for DNA extraction from cell culture medium, with some modifications to traditional DNA extraction protocols. Here are two commonly used methods:

Regardless of the method chosen, it's essential to consider the following tips for successful DNA extraction from cell culture medium:

The choice of method may depend on factors such as the available equipment, time constraints, and the downstream applications for the extracted DNA. Always follow the manufacturer's instructions or established protocols carefully to obtain reliable results.

assistant avatar

. مجموعة TriFECTa RNAi تستخدم لتقوم بصمم تحدبات RNA لتثبيط الجينات المحددة في الخلية. لاستخدام هذه المجموعة في الترانسفيكشن، يجب عليك أولاً تحضير ال

RNA

المستهدف باستخدامها.

لتحديد كمية وتركيز ال

RNA

المستهدف الذي يجب استخدامه، يعتمد ذلك على عوامل عدة مثل نوع الخلية المستهدفة والجين المراد تثبيطه وكفاءة عملية التحويل (transfection) الخاصة بك. عموماً، يمكن البدء بتجارب تجريبية باستخدام تراكيز مختلفة لـ RNA ومراقبة التأثير على تعبير الجين المستهدف.

بالإضافة إلى ذلك، من المهم أيضاً استخدام جهاز للتحكم في عملية الترانسفيكشن لضمان انتقال الRNA بشكل فعال إلى الخلية. يمكنك الاعتماد على العديد من جهوز الترانسفيكشن المتاحة مثل Lipofectamine أو Polyethylenimine.

The survival rate of cells after sorting depends mostly on three factors, the shear force created by the flow rate, the duration of the whole sorting process and the collection medium after cells been "shocked" with the given electric charge. In my years of sorting practice, I would suggest a 4 degree Celcius sorting for no more than an hour per sample, else increase the serum amount from 20-50%, just for cell recovery, increase also cell concentration to obtain target number faster, but do not exceed 1e7/mL.

Best.

When co-transfecting plasmids, the amount of transfection reagent to use can vary depending on several factors, including the transfection reagent used, the cell type, the size and concentration of plasmids, and the desired transfection efficiency. However, a common approach is to use a total amount of transfection reagent that is sufficient to effectively deliver the combined amount of DNA while minimizing cytotoxicity.

Here are some general guidelines for determining the amount of transfection reagent to use when co-transfecting plasmids:

By carefully titrating the amount of transfection reagent and optimizing the transfection conditions, you can achieve efficient co-transfection of plasmids while minimizing cytotoxicity and obtaining reliable experimental results.

l Check out this protocol list; it might provide additional insights for resolving the issue.

Lipofectamine 3000, like many other transfection reagents, can be sensitive to its expiration date. The expiration date is determined based on the stability of the components in the reagent, and using the reagent past this date can affect its performance and efficiency in transfection experiments.

Here are some reasons why Lipofectamine 3000 might be sensitive to its expiration date:

To ensure optimal transfection efficiency and reproducibility, it's important to use Lipofectamine 3000 within its specified shelf life and expiration date. Using expired reagents can lead to unreliable results and may necessitate repeating experiments, wasting time and resources.

Always check the expiration date of Lipofectamine 3000 before use and store the reagent according to the manufacturer's instructions to maximize its stability and performance. If the reagent has expired, it's best to obtain a fresh aliquot for your transfection experiments.

l Reviewing the protocols listed here may offer further guidance in addressing this issue

125ul optimem + 1,5ul 20microM RNAi

125ul optimem+2,5 ul RNAi max mix togheer and wait 20min RT

in the meantime replace the medium on your cells (i plate 2 10e5cells/2ml 6wells

with 1,25ml DMEM FBS witout ATbio

add the transfection medium overthe cells, mix..final volume 1,5ml analyse at 48 or 72Hours...work great (some people remove the medium after 2h and add complete media!)

work great see photo

Transfecting Raji cells efficiently can be achieved by optimizing several factors, including the choice of transfection reagent, cell density, and transfection conditions. Here's a general protocol along with some suggestions to enhance the efficiency of transfection in Raji cells:

By carefully optimizing these parameters and following the protocol, you can achieve efficient transfection of Raji cells for your experiments.

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

Hello Mansur Dabai Salisu

You may consider the following reasons.

1. The choice of which cell type to use for transfection is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent, choosing an appropriate cell type is necessary to maximize results. For instance, some cell types like MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake. Blood or immune cells that lack the proper endocytic machinery can minimize uptake and there are the macrophages that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents. So, select the right cell line for transfection.

2. Make sure that the cells are not 100% confluent (should be about 60-80% confluent) or in stationary phase at the time of transfection because actively dividing cells take up foreign nucleic acid better than quiescent cells.

3. Most cell types should be used between 4 and 25 passages for optimal transfection.

4. There could be a possibility that the DNA concentration may be too low, or DNA may be degraded. In such a case, you may increase the ratio of DNA :transfection reagent, and you may confirm DNA integrity by A260/A280 spectrophotometer reading (should be at least 1.7).

5. You may use serum-free media for DNA dilution because some serum proteins may interfere with complex formation. You may also increase complexing reaction incubation time.

6. Test the culture for contamination.

7. Do not use antibiotics at the time of transfection because cationic lipid reagents increase cell permeability. As a result they may also increase the amount of antibiotics delivered into the cells, causing cytotoxicity and low transfection efficiency.

8. Cells could suffer mechanical damage during the experimental steps. So, do not vortex or spin cells for extended period.

9. Some transfection reagents could be compromised due to improper storage.

Best.

You can add less serum to the culture medium (e.g. 2%)

But remember it might be the original measurement that was off. Lastly it might not matter, use the lower concentration as real. and It won’t hurt if there is a bit more than expected Unless you need to be very quantitative

You usually detect knock outs by knocking in a cassette with a selectable marker gene or cutting in two locations which causes a deletion large enough to be detected with PCR. Double check that your plasmids are using promoters appropriate for your organism. If you are simultaneously trying to knock in a marker then you can combine the system with single strand annealing proteins used for recombineering (lambda red for prokaryotes and ICP8 for eukaryotes).

Hi Nick,

Supplementation of media with biotin for serum-free culture of mammalian cells is a little complex by the fact that biotin is susceptible to oxidative stress and inactivation by UV radiation. Oxidation products of biotin are biotin sulfoxide and biotin sulfone. For more information on micronutrients in cell culture, you may want to refer to the article attached below.

The safe starting dosage for primary culture cells may be optimized as in cell culture media, the concentration range for biotin varies from 0.01 to 0.82 (μmol/L).

You may purchase biotin powder for use in cell culture from the link provided below.

Best.

it could be that the 293T cells may have more robust antiviral responses compared to Jurkat and Raji cells, leading to the inhibition of TTV replication. This could include the activation of interferon response pathways or the expression of restriction factors that limit viral replication.

@Hieu Nguyen

My negative control (water + qPCR mix) was expected to have the higest Cq but it had Cq similar to my samples at its highest dilution. This makes me think my water or master mix was contaminated and my titer was very low so it amplified the contaminant i guess.

For primers, I have been using primers for the ITR regions. They have been yielding reliable results till now.

If you have not yet tried adding protease inhibitors when preparing the protein lysate then that would be the first thing I would try. Secondly you might reduce the time after induction or reduce the temperature a bit more.

Lastly the level of these products is fairly minor and you might just ignore them.

However I would note that you DO NOT know if your protein is pure or not. Since you are doing a western blot by definition you are only seeing the proteins that are detected. You might wish to run a protein gel and stain to get an idea of the actual purity, if this is something important to you. But truthfully for many applications you do not need pure protein.

Can you please share the composition?

Hi Lauren Bracken

I see your plasmid size is high and that's why instead inserting GFP inside your plasmid (Not to exceed the sized more that can be linked with multiple issue even cell death) you are co-transfecting the cells with GFP plasmid.

Now, there is something missing in your question, how much of your plasmid you use along with how much of GFP concentration, here for sure you will use higher concentration of your blind plasmid and a little of GFP just to get a little signal of transfection. If this is the case, then 15 to 30% transfection is quite fine because your blind plasmid would be more in number as compare to the lower amount/concentration of GFP that is co-transfected with your plasmid, so ideally more of your plasmid would have been transfected as compare to lower amount of GFP plasmid.

If this is the case and let say, you use 1 ug of total both plasmid (100 ng GFP+900 ng Your plasmid) or (300 ng GFP + 700 ng Your Plasmid) in 24 well plate, just have a try to use the total amount of GFP plasmid alone to mimic your original condition, you will get good transfection because there will be only GFP (in total) that is responsible for the signal, and hence you can have a comparison justification for actual transfection (Your plasmid+GFP).

If you can let us know the amount of plasmid that you are using (Both your plasmid+GFP plasmid).

kind regards

Dear Esteemed Colleague,

Thank you for your inquiry regarding the use of Polyethylenimine (PEI) for cell transfection and lentivirus production. PEI is a cationic polymer widely utilized in biotechnology and research for the delivery of DNA or RNA into cells due to its efficiency and cost-effectiveness. Here, I provide a comprehensive guide on employing PEI for both transfection and lentiviral vector production:

Overview of PEI Transfection

PEI transfects cells by condensing DNA/RNA into nanoparticles through electrostatic interactions, facilitating the uptake of these nucleic acids by cells. Its high cationic charge density allows for effective complexation with negatively charged nucleic acids, promoting endosomal escape and enhancing gene delivery.

Preparation of PEI Solution

PEI Transfection Protocol

Lentivirus Production

Key Considerations

By following these guidelines, you can effectively employ PEI for both efficient cell transfection and lentivirus production. Should you have any further questions or require additional details, please feel free to reach out.

Best regards.

This list of protocols might help us better address the issue.

Dear Esteemed Colleague,

Thank you for reaching out with your query regarding the influence of plasmid size on the efficiency of transfection using Lipofectamine 3000. The question you've posed is essential for optimizing transfection protocols and ensuring the successful delivery of genetic material into cells. Let us explore this matter in a structured and detailed manner.

Lipofectamine 3000 is a widely utilized reagent in molecular biology for the transfection of nucleic acids into various types of cells. It has been engineered to enhance the delivery of plasmids, siRNA, and mRNA into both eukaryotic and prokaryotic cells. The efficiency of this transfection reagent, like others, can indeed be influenced by several factors, including the size of the plasmid DNA being transfected.

In summary, the size of the plasmid is an important factor that can influence the efficiency of Lipofectamine 3000-based transfection. Smaller plasmids generally exhibit higher transfection efficiencies, but with careful optimization, successful transfection of larger plasmids can also be achieved. It underscores the importance of considering plasmid size in the planning and execution of transfection experiments to ensure the reliability and reproducibility of the results.

I trust this detailed explanation sheds light on the complexities of plasmid size in relation to Lipofectamine 3000 transfection efficiency and assists in guiding your future experimental approaches.

Best regards.

This protocol list might provide further insights to address this issue.

You can transfect iPSCs with RNPs using a gentle lipofectamine transfection reagent. By this, the transfection efficiency will increase up to >97%. The next step is to single-cell sort the CRISPRed iPSCs on a 96-well plate in an enriched environment using Stemflex medium, cloneR supplement, and laminin-521. Using this combination you can achieve up to 70% clonal survival rate while ensuring they grow as homogenous clones. I discussed this in detail in a methodology paper here:

Contact me if you have further questions about the method.

To improve the transfection efficiency of K562 cells via electroporation, you can consider the following strategies:

By systematically optimizing these parameters and experimental conditions, you can enhance the transfection efficiency of K562 cells via electroporation. It may require iterative testing and adjustments to achieve optimal results.

Thanks for the information.

A few possibilities on why you are not seeing the signal after transduction:

1. The titer of your lenti is very low therefore very few/if any cells are being infected. Have you titered the lenti?

2. You are not using a high enough MOI. Typically transducing human cells requires an MOI of 5 to achieve >95% transduction efficiency.

3. You are attempting to induce expression with tet too soon after transduction. It takes a few days for the lenti to integrate into the genome. For instance, if you had mCherry under a CMV promotor, you would not see expression until about 4 days post transduction.

Regarding the transfection, since you are transfecting a plasmid you would see the signal much sooner since there is no integration into the genome.

The S2 kit comes with reagent for CaCl2 transfection. However, I used Cellfectin2 to transfect S2 cells and the efficiency is very high.

Narges Zargar-Balajam Thank you so much for your recommendation!

It is kind of tricky to troubleshoot this issue based on the limited information provided. You may want to label the lanes, such as "input", "IP", "IgG control", "exosome marker", and "Positive control", so people can understand the readout better.

Not sure if you use the homemade gels or purchased ones, but the rule of thumb is the pH of the gel may dramatically affect the protein electrophoresis. In addition, you may want to make sure the buffer is not too hot during the electrophoresis and transfer.

Good luck.

I would need to know what genes you had knocked out to make the cell line tumorigenic. It might be possible your cell line hit the Hayflick limit.

I would try other transfection vectors (TransIT, jetPei, ....) there is a lot of them, change the DNA/lipofectant ratio... but there is cells that never transfect well.... tried transduction with lentivirus ?....

Maybe you are sampling in a late time point, and the phosporilation occurs at early stage, try to do a time lapse experiment, as early as posible 0.5-2 h

Hello Rutuja Pendharkar

hrGFP fluoresces brighter in mammalian cells and is less toxic than GFP/eGFP, making it easier to use for mammalian expression experiments. It has a higher extinction coefficient and is more resistant to pH induced conformational changes as well as to solvents, detergents, and proteases.

However, in the paper attached below, use of hrGFP instead of GFP did not result in a larger proportion of transfected cells expressing fluorescence for extended period of time after transfection. Therefore, hrGFP does not appear to offer a significant advantage over GFP in transfection experiments.

Best.

Yes, the GFP signal can be detected by flow cytometry no matter if intracellular or on the surface.

Dear Stefanie

First, examine the efficacy of your DNA construct and transfection reagents in HEK cells. Let us know about the results.

Calculating the genome copy number of Adeno-Associated Virus (AAV) titer by quantitative PCR (qPCR) involves several key steps. This method is widely used due to its sensitivity and accuracy. Here is a detailed guide:

By carefully following these steps, you can accurately determine the genome copy number of AAV in your samples using qPCR. This method is highly sensitive and allows for precise quantification of the viral titer, which is crucial for many applications in gene therapy and virology research.

This protocol list might provide further insights to address this issue.

probably they will re adhere if you put them in a new plate at a lower density (and after separating the agregates by flushing up and down) but I am not sure that the transfected cells will reatach... to much work for an unsure result... HEK-FT are suppose to grow in suspension (F) and they adhere really poorly (you can improve adhesion by coating your plates with poly L lysine (or buy coated plate) but I would rather shift to HEK-T cells; they adhere much better on plastic (and even better on coated plates)

No, I haven't. But it is logically sound.

I would just use ~40% more µg of the 14kb plasmid, compared to the 10kb version....

Hi,

some expression vectors also carry sequences for in vitro cell-free translations. If your vector does not have it, try different one with these additional pieces. It will allow you to have your peptide or, at least, will show that problem with expression (on any level) in original setting. Good luck.

Hello,

An important and simple thing to try is to skip antibiotic when transfecting and/or the serum. In my cells, antibiotic-free condition made the biggest difference in survival but other colleagues have reported the serum or both.

If you have tested this in parallel with the other variables (time, lipofectamine reagent amount and siRNA amount), perhaps is best to change to a different transfection reagent, fugene is one of the best, but I usually use it for bigger DNA transfections, such as plasmids. For siRNAs I've used SAINT_siRNA transfection reagent, with 12h-24hour exposure and very little loss of viability and for me it worked better than lipofectamine 3000.

Ideally, at 40-50% confluency, if it's too high or too low it also interferes with viability and transfection efficiency. In my experience, optimizing using a 96 well plate is a bit tricky, and a larger format might help.

I would also recommend using a positive (like GAPDH) and negative/scramble control, to make sure the transfection efficiency is okay, control for off-target effects and to assess if perhaps the loss of viability is part of the knockdown of your target protein. In the last case, it's also important to then test with a different sequence of siRNA, to better control for off-target effects of targeted sirna.

Hope it helps ;)

Hope someone can answer this question. From a few pieces of literature I read, electroporation could transfect more linearized plasmid into cells in a short time, which indirectly increasing the DNA concentration in nuclear. Compared to this, chemical transfection needs 4-6 hours to intake those DNA. and the transfect efficiency of linearized DNA in chemical transfection is low. Also, you cannot transfect too much DNA by using chemical transfection, which may cause some immune resistance and then decrease the transfection efficiency. For transite transfection I don't know, both electroporation and chemical transfection could be used. I guess chemical transfectin kit is much more easier to approach, and the procedure is more easier to follow.

1. Depends on what is in the plasmid you are using. If your plasmid is using some transposons then you can get almost random integration. The copy number will depend on if it is a jumping or replicating transposon. Viral vector derived plasmids tend to integrate into specific regions (eg. Lentivirus derived ones) but can be multiple copy. If integrated using recombinases (like the CRE/Lox system) then insertion will be site specific and usually single copy depending on the number of landing sites and if the genome is diploid. Similarly plasmids that integrate by homologous recombination will have 1-2 copies. The part of the plasmid integrated is usually a specific cassette flanked by regions used for integration (eg. homology arms, recombination sites or ITRs). Most plasmids try to avoid backbone integration into the host genome as that is prone to gene silencing by methylation.

2. You are correct everything between the LTRs is integrated. You will find most plasmids of that variety have a cassette with your GOI and the selection marker that is flanked by the LTRs with the replication machinery on a separate helper plasmid.

3. Your GOI and selection marker should be integrated together. After transfection treat each clone as a separate cell line and maintain selection for a least a month. It is good to periodically screen for the marker as that will eliminate any clones that have had the integrated cassette silenced.

4. I would have to look the maps of those specific vectors.

5. Yes the probabilities are combinatorial to an extent though with enough different construct being co-transfected there would eventually be signs of toxicity.

Hi,

You can fix your cells with PFA (room temperature) or ice-cold methanol (4 C) for 15 minutes, then rinse with PBS 2-3 times. Cells can be left in PBS in the fridge and this won't affect fluorescence. When you are ready you can resume the experiment starting with the permeabilization/blocking step (with triton and BSA).

Worked all the time for me

Hope it helps

Choose a plasmid with fluorescence and test it on 293 cells. You can see if it is inactivated.

You can start with these papers as reference.

You can use the same ratio as recommended by the manufacturer for siRNA. At the end of the day it is a matter of phosphodiester charged versus cationic lipids...so you should be OK. Best

Kajs Hadžić

Thanks so much Kajs. It's really an open-ended question and your insights are very valuable :)

Thank all of you very much for answers. I tried to optimize (amount of DNA, T7E1 concentration, incubation time) but i observed overdigestion. Our final decision is to send sanger sequencing.

Hello Dmitry Lifanov ,

I think it depends on more than one factor, including the characteristics of your cell line, your plasmid, the concentration of G418 that your cells are resistant to ...

Maybe if you can, try to cultivate the transfected cells with and without G418 (in parallel) for ~5 passages : you might see a difference if the cells lose the plasmid, if you can test for the presence/absence of your gene of interest after this culture.

If the cells don't lose the plasmid after 5 passages, maybe you can go up to 10 passages or more. It depends on how long you will need to co-cultivate them with the non-resistant cell lines.

I hope I could help a bit ! Good luck with your experiments.

The plasmids do not "connect" to each other in the host cell, they're just co-expressed in the cell which means all the different components needed to make the lentiviral particle are expressed in the cell.

This improves safety because the genome of the lentiviral particle that is produced in this way will only contain the transgene from your transfer plasmid*, since that's the only plasmid containing LTRs which are required for the RNA genome to get packaged into the lentiviral particle. The lentiviral particle won't contain the envelope genes or packaging genes (which are on the other plasmids), and those genes are necessary for making the lentiviral particle. So the only time the lentiviral particle can be made is when you co-transfect all three of those plasmids. Once you isolate the lentivirus and then use it to infect other cells, it won't be able to replicate because it doesn't express the necessary genes.

And for your last question, it is completely normal to test overexpression of a gene by integrating copies into the genome with lentivirus. This is very common. But it's important to keep in mind that your gene of interest may behave differently because it will be expressed at different levels than endogenously.

*not completely true but I'm simplifying things.