Shen et al (Nature Methods, 2017) explore and identify synthetic interactions among 73 cancer-associated genes. To perform their loss of function screen they combined CAS9-expressing cell lines with a sgRNA library of high titer lentiviral particles. Most of these gene interactions were subsequently validated by drug treatment. [Read more…]
We previously addressed the PD-1:PD-L1/PD-L2 immune inhibition and the corresponding cell-based tools to screen immuno-oncology agents for restoring antitumor immune response. Unfortunately cancer cells have other ways to escape and survive. Let look together at another immune inhibition pathway, namely the TIGIT:CD155/CD112.
As messenger RNAs (mRNAs) are easier to deliver into cells then plasmids or viral vectors, they are useful for non-dividing cells. Inversely to the vectors, they ensure genome integrity that is recommended for cell therapeutics. mRNA are also well adapted to transient expression as required for cellular reprogramming, gene editing, and vaccines.
You might like to take a look at this poster, presented by Trilink (renowned modified nucleic acid experts) at the Keystone symposia last March (Pattern Recognition Signaling: From Innate Immunity to Inflammatory Disease). They share results on how to optimize messenger RNA for therapeutic activity.
“Innate Immune focused approaches to maximize messenger RNA therapeutic activity”
Download your copy of the poster here.
The HiP™ (High Purity) distinction by BPS Bioscience starts, of course (as the name says), with a high purity level. But that’s not enough. Such pure proteins may aggregate, which is not compatible with binding assays. Thus, the HiP™ label also demands a low level of aggregation, or even none at all. [Read more…]
PD-1 / NFAT Reporter – Jurkat Cell Line
The PD-1 reporter cell line is a T cell line expressing luciferase under the control of NFAT response elements. The level of the luciferase activity measured with the One-Step luciferase detection system (BPS Bioscience) corresponds to the T cell activation in response to the TCR activator from the target cell.
To escape, the target cell may present PD-L1 or PD-L2 to PD-1. Indeed, the interaction inhibits the T cell activation. It leads to decrease in luciferase activity. [Read more…]
DNA is a useful source of information for research and medicine. It starts by collection, following which it is deciphered by a wide range of analysis, from simple genotyping to deep whole genome sequencing.
The most popular way to collect DNA is drawing blood. Not very pleasant for the patient, as we have all noticed, and it also requires an experienced person, a nurse. The blood must be kept cold and DNA extraction should be done not long after the sampling. Still, it brings high yields of up to 30µg od DNA.
Alternatively, saliva sampling can be used to collection DNA. Sampling is painless, but it is still not an obvious choice especially with children. Yields are highly variable, and lower than with blood. Furthermore, kits are quite expensive.
Today, there is a sampling method to easily collect DNA, whilst also providing high yields. It is based on buccal sampling. I invite you to take a look at how it works and the benefits for your studies. [Read more…]
We need to find biomarkers for prognostic, diagnostic and personalised treatment development. Notably to fight cancers that affect tissues. Since biopsies are invasive, it’s better to look for biomarkers in body fluids. Indeed, a simple blood sample becomes a kind of ‘liquid biopsy’ to reveal tissues affections. For 13 years, increasing interest has been shown for miRNA as biomarkers and it will last for sure. The 2 main reasons are that they are major regulators of cell processes and they are released from tissues into the blood. They are major biomarker candidates in serum and plasma. Thus, these circulating miRNA (cmiRNA) are the best hope for modern medicine. Still, a lot of research has to be done to determine the specific signature for each pathology, and also depending on the patient background. Obviously, cmiRNA profiling is a key step and requires sensitive and reproducible method. Sequencing, qRT-PCR, several kind of microarrays… Let’s explore together what the best approach could be. [Read more…]
The main challenge when choosing a transfection reagent is that we don’t know how it will work with our own cell type of interest. It is also time consuming to find the optimal conditions. Well, here’s the solution: pre-optimised transfection reagents.
They already cover more than 39 cell types including MEF, Caco-2, MCF-7, HepG2, Primary Macrophages, Huh-7 and many others.
Sharing our feedback on performances
Using a vector expressing the eGFP (pEGFP-N3) under CMV promotor, we assessed the transfection efficiency. Take a look at the example of results below. They will give you a pretty good idea of what you can expect.
Can we compare the pre-optimised Genjet?
Ok, now you can see we got good feedback. Still, will it be a better solution?
The answer is yes. The comparison with Lipofectamines, Fugene HD and Amaxa reveals that pre-optimised Genjet reagents allow high number of positive cells (dark green below).
You can see on right of each picture just above, that performances are even better with 10% serum, that would be much appreciated by the cells.
Furthermore, this quality is associated to low price. Check it for yourself!
In March 2016, Mark J Osborn et al published in Molecular Therapy a major article for genome editing (doi:10.1038/mt.2015.197), about knock-out of CD3 in human T-cells. The goal is to improve T-cell-based immunotherapies to fight tumours using engineered allogenic T-cells from healthy donors. It is a very good example of how CRISPR-CAS9 can help medecine. And even if you are not very comfortable with CAR T-cells and the treatments of malignancies, I would recommend you read it and especially take a look at figure 2. Indeed, dear friends of genome editing, the authors made a clear and fair comparison of several KO strategies, covering all the main options. Thus, it is not only a major step for anti-tumour treatments but it is also an excellent overview that reveals the best approaches. So, before reading this post any further, you might like to read the article mentioned above. [Read more…]
Stable expression in a cell lines is very useful for numerous projects. For example, it helps in Eukaryotes for optimization of protein productions and it is convenient for recurrent needs and high yields. Stable exogenous expression of tagged protein allows extensive tracking and localisation of the protein in live. Cell lines stably expressing a protein can be used for various screenings such as lost of function assays (CAS9-expressing cells), and reporter-based assays (Gaussian luciferase). Of course, the stable expression could also be shRNA for knock-down.
Stable expression implies insertion into the genome. It used to be random and could cause hits with side effects. Now, targeted insertion in Safe-Harbor sites is now possible. How can you take advantage of this progress?
Mouse ROSA26 and Human AAVS1 safe harbor sites
First of all, what is a safe-harbor site? It is an ideal site in the genome in which we can add a construct without harm and expect consistent level expression. For years, researchers have looked for it. In 1997, Zambrowicz et al initiated the discovery of the ROSA26 site on Mouse chromosome 6. It was shown it is a transcriptionally active region with an open chromatin configuration and transgene insertion has no or minimal effect on global and local gene expression. Remarkably, a ROSA26 inserted transgene is expressed in all tissues.
Similarly, in the human genome there is a safe-harbor site on chromosome 19 (locus PPP1R12C) called AAVS1. It was described more recently by DeKelver, et al. (2010). It has become a remarkable safe harbor site for ESC (Embryonic Stem Cells) and iPSC (induced Pluripotent Stem Cells) because of the robust expression of harboring constructs and the absence of abnormalities or differentiation deficits.
Since absence of visible effect doesn’t mean there is no at all risk of effect, I should mention that an ideal genomic safe-harbor doesn’t exist yet. Nevertheless, AAVS1 in human and ROSA26 are certainly the safer harbor sites today.
Comprehensive Safe-Harbor kit
Today we can easily insert a construction into a targeted site of the genome and so maintain its integrity. The principle is based on the targeted insertion of a Donor construction as illustrated in figure 1.
Each kit includes vectors expressing the system (CRISPR-CAS9) and the following:
- Donor vector in which you clone your ORF of interest
- RFP Donor control to monitor in fluorescence the knock-in
- Primer pairs for the PCR analysis of the genome integration
You can also obtain the Donor vector with your ORF of interest upon request.
The RFP control is highly convenient allowing a direct monitoring of the transgene genome integration (Figure 2).
Comparing with the control (without Donor), we can see the high integration efficiency after only 12 days of puromycin selection.
Comparing CRISPR-CAS9 Safe-Harbor to the classic method
Well… Think easier, faster, more reliable and cleaner!
So, is it a new way to work? As soon as you get the Safe-Harbor kit (with the empty Donor vector), you can use it indefinitely to establish promptly isogenic and polyclonal cell lines expressing all the ORFs you need. And furthermore the cell lines will be more reliable than random integration of lentivirus or plasmid.
Any questions? Please feel free to get in contact by leaving your comments below!