The goal of this protocol is to simplify CRISPR/Cas9 mediated knock-in experiment in macrophage and T-cell lines, with the aid of fluorescent reporters and affect cell sorting. ROSA26 Locus is known as a genomic safe harbor site for insertion of transgenes. It is a usual to express protein of interest with or without attack in the mouse world of 26 locus, or in the human ortho log.
Part 1:Design and the Plasmid Construction of sgRNAs Targeting Rosa26 Locus. First, design sgRNAs from mouse Rosa26 locus, using sequences from mouse genome informatics, and Ensemble Genome Browser, then download the genomic sequence of the mouse Rosa26 gene. Go to the online web tool, CRISPOR, paste 100 base pair of the input sequence from the Rosa26 Locus.
Then select a genome, for example, Mus musculus-mouse reference genome 2011. Next, select the type of PAM. Use 20bp NGG"PAM motif, recognized by spCas9.
Click Submit. Choose guide with a high specificity score, high doing score, and a fewer off targets. Guides indicated as inefficient should be avoided.
Notably, it's preferred to select a two guides with overlapping sequences, which may increase the knock-in efficiency as reported. For warm dine sequence, synthesize a forward illegal and a reversal illegal, which annealed phosphorated and ready for cloning to factor. Prepare the linearized decrease per Vactor to which could express is I have to reporter by BBS one digestion, like get the Neal, the legals to the linearized vector to generate the final construct, which simultaneously expresses guide RNA and the CAS nine nucleus linked in with a DSRIP two fluorescent reporter.
Part Two:Construction of Targeting Vector as Homologous Recombination Template. For expression of the protein of interest. For example, the human lasted up.
You want synthesize the cDNA sequence by commercial source and affinity OST tag or other commonly used attack for purification of protein can be fused to the cDNA sequence. Remember to include the Kazak consensus sequence before the initiation of the cDNA. Digested by Asc1 restriction enzyme, and like get the CD and the insert into the backbone.
Vactor, namely pKhR26-IBFP IBP yielding the final targeting concentrate the container, each one, KB of five prime and three prime homologous arms. The expression cast set includes a CG promoter, the UST restaurant, if you want Koonin region linked to an Iris, if VFD reporter and a poly AC funds. Finally on horizon targeting vector using unique cutter enzymes, such as EcoR1 or BamH1, the digests and product is purified in a store that minus 20 degree onto electroporation notes.
It is recommended to obtain high concentration of linearized vector DNA. Part three:Electroporation of Macrophage and T-cell lines prepare cell culture followed separation for Geritalk cells. Optimal cell density was approximately 0.5 million cells per ML at the time of transfection.
Full electroporation in ten macro little nuclear fashion tip formats prepare a 24 well plate containing 500 microliter of complete growth, medium without antibiotics in each well. Prewarm the plates in humidified 37 degree, 5%covering that setting incubator. Turn down the electric operation system input electroporation parameters for jet cat or Rosales.
Prepare cell DNA mixture follow-ups or operation collect chicken cells. Curchin 25 square centimeter flask transfer cells in the 15 animals. Since you tube, then Phillip the cells by centrofugation at 90 times G for eight minutes at room temperature, ask for it, the supernatants.
For washing, Resuspend the cells with the five ML PBS, then spin the cell suspension by centrifugation using the same condition as the previous step. Remove the DPPs. And resuspend the cell pillows using two ML of DBS.
You stay micro little cell suspension and mix with the equal volume of 0.2%Trepan Ballou to estimate the cell count load sample in terms counting slot insert Connie slide in the automated cell Connor and view the image themselves. Then get a cell count result. Calculate 2 million cells will find reputations of electroporation per knocking experiment transfer the number of cells into 1.5 ML.Centrifuge two pellet cells by centrifugalization.
To save time, electroporation mixture can be prepared during the centrifugalization that add 2.5 microgram of each of CRISPR CAS nine vector 2.4 microgram of Belinda rice, talkative vector, and re suspension Buffalo. Our total total volume of 55 macro litter in a new 1.5 ML centrifuged tube gently vertex and. Briefly stand to mix well.
Leave the Luxor pressure mixture and room temperature before use. After spinning aspart, the DTB is this essentially fuse for an additional 30 seconds, remove as much supernatant as possible re suspend cells by adding the prepared electroporation mixture pipette up and down gently. Electroporation asked for the cell electroporation mixer using 10 micro little nuclear faction tip was pipettes important.
Avoid air bubbles during pi petting it'll cost electroplating failure. Press start. after which the operation transfer the sample in a while.
Well of 24, well plates with a pre-warn the gross medium perform the same electric pressure and procedures as above on the remaining four replicated samples, gently rock the plate to assure even distribution of the cells. Incubate the cells in 37 degree incubator for 48 to 72 hour. Prior to fact, cell sorting At 24 hour post transfection, examine the expression of disrupt two for restaurant reporter using florescent microscopy detached from the rep for us themselves indicates that the electroporation is working properly.
Part 4:cell sorting to isolate putative knock-in cells. Before cell sorting prepare, in 96 well plates with 150 micro little of cell type specific gross medium per well use wrong bottom micro place for culture and itself and the flat bottom micro place for all cells transfer cells into a sterile FACS to keep the tube in on ice and to put the box in the path through window, connecting to cell sorting room when applicable set cell sorter with 85 micro meter nozzle and low flow rate for immune cell sorting to achieve optimal cell evaluability and survival take samples from the pass-through window vertex briefly, and load the sample in the acquisitions. Champer set up fact skating for cell sorting first defined sales funnels for Ford scatter and side scatter, then define the life cells by gaining the set talks red negative cells.
Next, they find a live single cells by single singlet gating using the FSC H versus FSE a multi-varied block. Finally set a gate for the desired DSRs two and B if the Volvo positive subpopulation, which indicates cells successfully co transfected with CRISPR vector and the pocketing vector. So 10 single cells in the each well of the 96, well plate supplemented with the prewarm, the complete growth medium using period mode or single cell mode after sorting incubate the 96, well plates in the cell culture incubator for cell expansion.
Part five:screening of elevation of positive knock-in cells After approximately two weeks of cell expansion past the survive, the cells in the 48 well plate assess the expression of BFP or flow cytometry using a small proportion of proliferate itself using the wall type cells as a negative control cell populations, possessing higher percentage of BFP positive cells are kept to fulfill the validation extract the genomic DNA from the cells we should express BFP pass the isolated genomic DNA by PCR with primers spanning each side of the homologous arms, namely one primary locates in the genomic sequence, exterior to the targeting vector. The seen side to talking to the vector in this experiment, the predicted the size of PCR product amplified with the primary is 1.2 KV.And with F two and R two is 1.2 KB Sanger sequencing results. So PCR products show the sequences of each integration junction demonstrating the correct and knocking position into the Rosa 26 Lucas.
I says like immuno blotting enables for the validation of correct knocking at the protein level part six representative results. As an example, published in previous study, we expressed that get 45 molecules tack that the inter with an OSD tag, and there was a 26 locus in checkout T-cells subsequently USD tact. I get 45 and incident directors or pulled down by affinity purification and a subject to mass spectrometry.
They proteomics data revealed interaction of T-cells. Therefore this product pool has greatly facilitated the search for novel intercalators to elucidate the function of a given protein. This technical presentation showed that by transiently expressed increased per cast line RFP with the talking vector co-expressed and BFP for Rosa 26 locus.
We generated knocking cell lines with permanent gene expression in both T cells and macrophages, which are difficult to perform in genetic manipulations. This methods and recommendations are applicable to Chris permitted in assertions of large DNA segment for mechanistic studies of immune cells, such as for identification of protein, protein interaction study.
