The visualisation of proteins or organelles in cells and other complex biological systems is ‘bread and butter’ stuff for cellular and molecular biologists; it’s performed day-in, day-out in labs across the globe. But this doesn’t mean that the most popular approaches currently used for protein visualisation – dye staining, antibody labelling and fusion protein over-expression systems – are ideal. Almost every scientific technique has its advantages and drawbacks, and the various options currently available for protein visualisation are not exceptions to this rule, so let’s have a look at the pros and cons of each.
Recycling has always been a smart idea, and nature has its own processes to ensure that waste is kept to a minimum. As Professor Ohsumi discovered, autophagy is the cells way of degrading and recycling cellular components, allowing it to adapt to nutritional deficiency or other environmental influences. Professor Yoshinori Ohsumi, honorary professor and leader of the Cell Biology Unit at the Tokyo Institute of Technology, has been studying autophagy for 27 years. This year's Nobel Laureate discovered and elucidated mechanisms underlying autophagy, according to the Press Release from The Nobel Assembly at Karolinska Institutet.
The CRISPR/Cas9 system has been rapidly adapted to practically every model system for its ease to generate and high efficiencies to cleave target DNA. But unlike our experience with Zinc Finger Nucleases, in the human, rat and mouse cell lines we tried successful co-transfection of Cas9 mRNA and sgRNA was cell-line dependent, and often resulted in either very low or no cleavage activities.
However, sequential transfection of cells with Cas9 DNA first, and sgRNA followed 24 hrs later, reliably produced good level of activity, indicating the requirement of Cas9 presence at the time of introduction of sgRNA. Not surprisingly, creation of a cell line stably expressing Cas9 led to consistently high cleavage activities upon transfection of sgRNAs. Transfection of recombinant Cas9 protein pre-complexed with sgRNA (ribonucleoprotein particles, or RNPs) led to efficient cleavage as well.
On the other hand, when Cas9 mRNA and sgRNAs are co-microinjected into single cell embryos, it produces target cleavage as efficiently as RNPs to produce straight KOs and large deletions between two target sites, again raising a question of local concentrations of Cas9 protein and sgRNA.
Below we summarize some of the work we've done optimizing delivery of CRISPR-Cas9, which which can be read in full publication form in Human Gene Therapy here.
Here you'll find a complete list of our most frequently asked questions relating to X-MAN cell lines. How are they made, how are they validated, and how can you use them? Read on to find out.
Whether it's a stock you've expanded following it's arrival from the cell bank, or a clone you've carefully nurtured from the single cell, banking down your cells in the right way is crucial if you're going to be able to return to them time and again, and revive them quickly so that you can get on with your experiments.
Here's our protocol for banking human cell lines:
Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.
If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:
Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.
We have combined our proprietary genome editing technology with large scale RNAi screening capabilities to allow identification of novel drug targets.