CRISPR genome editing: which cell line to choose?

Many labs have adopted the CRISPR genome editing technology to make knock-out and knock-in cell lines.

This technology produces first a targeted break in genomic DNA, which can then be exploited to produce cell lines with genes knocked out or where a donor vector has been used to introduce new genetic elements (point mutants, fluorescent tags, antibiotic resistance cassettes, etc.). Essentially any desired modification to the cells genome can be made. In setting up these genome editing projects there are many choices to be made including vector for the Cas9 protein and for the sgRNAs. Perhaps the most difficult choice, however, can be which cell line to use. Even the most affordable stable genome editing cell line development services can come with a significant cost, so choosing the right cell line at the beginning is crucial. Here we explain some of the choices researchers have in setting up their CRISPR genome editing projects and give our advice for cell line selection.

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What’s new in qPCR?

We know molecular biologists spent a lot of time setting up their qPCR protocols and aren’t about to change the way they do things. We also know they are technically savvy and like keeping up with the recent advances, so we prepared this brief update about what’s new in the world of qPCR.

Molecular biologists know that in qPCR, you generally have a choice between the dye-based (e.g. SYBR® Green) protocols and the hydrolysis (e.g. TaqMan® MGB) probes. Most researchers have a general impression that it takes a good amount of expertise to set up good qPCR assays with a dye-based approach and that, in the hands of an expert, a SYBR® Green approach is the only way to have truly quantitative PCR. The hydrolysis probe-based approach is thought to be more end-user friendly and will yield reasonably trustworthy results even if you don’t have the world’s expert on qPCR working in your lab and is also the best for multiplexing. The thing that both qPCR methods have in common is that users are generally fairly loyal to their technique, so people tend to have ignored the innovations that have occurred in the field of qPCR over the past several years. Here, we’ll try to bring you a brief history of the past few years of qPCR innovation. [Read more…]

Chemically synthesized mRNAs: now a reality

In vitro transcription has been a common protocol in RNA biology laboratories wishing to work directly with mRNA molecules to study phenomena such as mRNA translation. Commercially available kits have greatly facilitated the capping and polyadenylation and in vitro transcription of RNAs, but now there is another option:  ordering chemically synthesized mRNAs.

In vitro transcription kits such as the T7-FlashScribe™ Transcription Kit allow maximum RNA yields in 30 minutes. Subsequent processing of primary transcripts involves addition of the 5′ cap and 3′ poly(A) tail.

N-7001

m7GTP mRNA cap

N-7003

ARCA mRNA cap

Some of the most popular kits for mRNA processing include the ScriptCap™ m7G Capping System for capping and the A-Plus™ Poly(A) Polymerase Tailing Kit for tailing. Researchers wishing to optimize protein expression use chemically-modified mRNAs, such as those carrying an anti-reverse cap analog ARCA at the 5′ end. Again, kits such as the MessageMAX™ T7 ARCA-Capped Message Transcription Kit have made in vitro transcription of ARCA capped mRNAs routine in laboratories. When a standard m7 GTP cap is added to mRNAs in vitro, only about 1/2 of the cap is added in the correct orientation. The ARCA cap is modified with a methyl group to prevent capping in the “incorrect” orientation, thus resulting in a higher percentage of efficiently translated mRNA.

pseudouridine

Depending on the application, researchers are seeing advantages of using other chemical modifications as well. Pseudouridine-5′-Triphosphate, a naturally occurring base, is used to decrease nuclease activity and TLR activation, and modified cytidine base (5-Methylcytidine-5′-Triphosphate) is used for similar reasons. Warren et al. 2010 (doi: 10.1016/j.stem.2010.08.012), for example, found that mRNAs carrying an ARCA cap and Pseudo-uridine and methyl-cytidine substitutions are very efficient for reprogramming of many human cell types and fail to activate the toll-like receptor innate immune pathways.
Similarly, the Immune Stimulation Transcription Nucleotide Set is available for those who want to purchase ARCA cap, Pseudouridine-5′-Triphosphate, and 5-Methylcytidine-5′-Triphosphate. Another modification gaining popularity is the modified uridine (2-Thiouridine-5′-Triphosphate), however this popularity may be a result only of intellectual property limiting the commercial use of Pseudo-uridine.

EGFPmRNA_expression

Commercially-available capped and polyadenylated mRNAs modified with pseudouridine and 5-methylcytidine include those encoding:

Gene
Pseudouridine
5-methylcytidine
Length (nucleotides)
EGFP mRNA (5meC, Ψ) + + 996
Oct4 mRNA (5meC, Ψ) + + 1,359
Klf4 mRNA (5meC, Ψ) + + 1,688
SOX2 mRNA (5meC, Ψ) + +  1,230
c-Myc mRNA (5meC, Ψ) + + 1,596
Lin28 mRNA (5meC, Ψ) + + 906
FLuc mRNA (5meC, Ψ) + + 1‚929
NLS-Cre mRNA (5meC, Ψ) + +  1‚350
β-gal mRNA (5meC, Ψ) + +  3,336
Factor IX mRNA (5meC, Ψ) + + 1,662
hAAT mRNA (5meC, Ψ) + + 1,530
mCherry mRNA (5meC, Ψ) + + 996
Eira CFP mRNA (5meC, Ψ) + +  978
Blaze YFP mRNA (5meC, Ψ) + + 987
Cas9 Nickase mRNA (5meC, Ψ) + + 4,341
EPO mRNA (5meC, Ψ) + + 858
CD8 mRNA (5meC, Ψ) + + coming soon
NGFR mRNA (5meC, Ψ) + + coming soon
Guassia Luciferase mRNA (5meC, Ψ) + + 834
Renilla Luciferase mRNA (5meC, Ψ) + + 1,212
Cas9 mRNA (5meC, Ψ) + + 4,509
EGFP mRNA (5meC) + 996
FLuc mRNA (5meC) + 1‚929
OVA mRNA (5meC, Ψ) + + 1,437
Cas9 mRNA (Ψ) + 4,509
EGFP mRNA 996
FLuc mRNA 1‚929
β-gal mRNA 3,336
OVA mRNA 1,437
Cyanine 5 FLuc mRNA (5meC, Ψ) + + 1‚929
Cyanine 5 EGFP mRNA (5meC, Ψ) + + 996

 

Clearly, many of these purified mRNAs encoding fluorescent proteins, luciferase, or other reporter proteins are intended as controls for users setting up assays on which disease-relevant mRNAs will be tested. Once the assays are established, users now have the choice to produce their mRNAs of interest themselves using the in vitro transcription, capping, and polyadenylation kits described above or to order high quality mRNAs produced by expert chemists. Chemically synthesized mRNAs can be synthesized to contain nearly any sequence and chemical modification desired and gram quantity yields are possible. For particularly long mRNAs (up to multiple kilobases), in vitro transcription steps may still be required, but experts at TriLink Biotechnologies are able to design custom strategies to optimize yield even for the most complicated custom mRNA production requirements.

European scientists interested in learning more about out-sourcing mRNA production are encouraged to contact the local TriLink Biotechnologies distributor, tebu-bio.

mRNA delivery tools

Direct delivery of RNA sequences to a cell circumvents many drawbacks inherent to plasmid or viral DNA. This innocuous strategy reveals being as efficient as viruses when it comes to conveying and expressing nucleic acid sequences in non-dividing cells, for it does not rely on nuclear entry, precluding any mutagenic events by the same token.

Once efficiently engineered to escape their automatic and swift destruction in most biological environments, RNA molecules become remarkably stable and turn out to be extremely reliable for in vivo applications. In line with our Nucleic acid delivery tools presentation series, let’s focus here on RNA delivery to the cell. [Read more…]

New 3D-gene® miRNA and mRNA expression profiling service

Recently, tebu-bio and Toray reached agreement for miRNA and mRNA profiling lab services. Already recognised as a European service provider in genomics, proteomics and cell-based assays, tebu-bio now brings European researchers access to Toray’s 3D-Gene® microRNA and mRNA profiling technology, offered through lab services.

During recent lab collaboration sessions, Toray’s 3D-gene® experts and tebu-bio’s lab staff shared their experiences and skills regarding miR and mRNA profiling technologies. A nice opportunity for me to ask Hideo Akiyama, PhD (Deputy General Manager – New Projects Development Division at Toray Industries, Inc.) 3 questions regarding the power of this “3D-gene® black resin” for microarray analysis.

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microRNAs: Keys to open cell biology’s secret chamber

As cell biologist, when working in labs I have in the past witnessed several “X-files” in a flask of cells along my way: all cells of an established cell line are supposed to have the same genetic background and to respond similarly to the same stimuli… but sometimes this is simply not what happens!
I would not risk saying that it all comes down to epigenetics, but much of this “unexpected” behaviour is governed at  mRNA level independently of the genetic background. “Cell personality” is then defined at several levels: genetic background, epigenetics, mRNA modulation and protein/lipid content.

It’s a sophisticated web of positive/negative signals that governs cell fate and behavior. But how do cells cope with this complicated issue? [Read more…]

3 good reasons to select capped polyadenylated mRNA expressing factors for iPSC generation

mRNAs are expression factors that mimic fully processed mRNA. Being the substrate for translation by ribosomes, mRNA expression factors are often preferred over viral vectors for cell reprogramming and iPS cell generation because of the absent risk of integration into the genome. Such RNA-induced pluripotent stem cells (RiPSCs described in 2010 by Warren et al.) are becoming more and more popular. 3 reasons might illustrate RiPSCs’ attractivty.

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