I would like to know the detailed process of KI cell line construction. Could you please provide me with the specific transfection method and monoclonal selection method for this experiment? Thank you!
The question is vague and elusive.
You need to provide more information about the KI cells you want to achieve.
For example, in human iPS cells, you want to insert EGFP gene or drug resistance gene to KO XX gene.
Whether it is microbial experiments, animal cultured cells, embryo manipulation in fertilized eggs, or genetically modified plant production, you should be more explicit in your questions.
Thank you very much for your attention to this question. I apologize for my lack of clarity.What I really want to know is how to achieve transfection and selection cloning during the construction of KI cell lines?For example, is it transfected with a lentiviral vector or a non-viral vector?Are Cas9 plasmid and donor plasmid transfected in two steps or co-transfected?How to screen and select monoclones after transfection?Selecting under microscope or by flow cytometry? @Narumi Uno
I often do Knock out and HDR in human iPS cells. The following vectors are recommended for CRISPR / Cas9 expression. PX330-U6-Chimeric_BB-CBh-hSpCas9 provided by Zhang lab. You can purchase it from addgene. Since pX330 is not highly expressed in human iPS cells, Knock out is easy, but the frequency of homologous recombination is low. I have the impression that the Cas9 expression system by the EF1 promoter is superior.
Please refer to this paper.
https://doi.org/10.1016/j.ymeth.2015.10.015
On the other hand, in recent years,
Cas9 and gRNA can be purchased directly as purified proteins and synthetic RNAs. I purchase from IDT (integrated DNA technology). Although expensive, they are simple and very efficient. It's a lot cheaper than making mistakes. Numerous detailed protocols are provided.
The HDR vector has homologous recombination sequences (about 1 kb) corresponding to the cut site(s) on both ends, and drug resistance markers and fluorescent markers are placed between them. An EF1 promoter or CAG promoter should be used to express the marker protein. The CMV promoter will easily disappear.
These are examples of introduction.
gRNA expression pX330 vector: 2µg
Circular fluorescent protein expressing-HDR vector: 8 µg
HiPS cells (Feeder-free cultured): 3x10 ^ 6 cells
NEPA21 electroporater was used for gene-introduction.
It is better to use the HDR vector as it is in circular preventing random integration of the HDR vector. Fluorescence expression will be observed from the next day. Transient fluorescence expression lasts for several days. It is possible to acquire DNA 48 hours after gene transfer and evaluate whether homologous recombination has occurred by PCR analysis. Fluorescence-positive cells are obtained by FACS one week later in order to obtain cells with homologous recombination.
The obtained cells were seeded onto 200 cells/384-plates.
If drug selection is possible, the electroporated (3x10^6cells) cells would be seeded onto three of 10 cm dishes and selected by antibiotics for 2-weeks.
These may be very rudimentary methods, but they are commonly used.
Do you have any questions?
Best,
Narumi
I really appreciate your good advice and this review. I have read it carefully.Now I have another question to ask: if I want to insert a GFP reporter gene into the target gene to facilitate subsequent observation of the expression and localization of the target gene, should I screen heterozygous clone or homozygous clone?@Narumi Uno
Jing Yang
In general, heterozygous clones are often used to ensure the normality of the cells. In homozygous clones, normal phenotypes may not be observed due to loss of functionality of the target gene by knock-in.
He has a point. I think homozygous knock-in tends to be more bright. In the end, I think it depends on what you're looking for.
If you try to get a homozygous clone by genome editing, you should also get a heterozygous clone in the same trial. I think you should evaluate both and choose the one that satisfies you.
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