MNCs/ MKs [https://www.dreamstime.com/]
Monocytes (MNCs) play an important role in immune defense, inflammation, and homeostasis by sensing their local environment, clearing pathogens and dead cells, and initiating adaptive immunity, as well as providing a progenitor pool that contributes to inflammatory DCs and replenishes some tissue macrophages. MNCs are a heterogeneous and plastic cell population. It’s subset function has been more extensively studied in mouse models than in humans.
mo-DCs_ IL-4, GM-CSF [mainly by CD14++CD16– subset]
M1 macrophage_ GM-CSF [all subsets]
M2 macrophage_ M-CSF [all subsets]
Functional observations of human monocyte subsets documented in the literature are somewhat controversial, with different responses observed depending on the context of their activation.
“Model Animal Research Center of Nanjing University”
Those mice models were established as myelo-monocytic cell tracing system, were developed for study the development of monocytes in vivo by MARCNU. Ms4a3 is a gene that specifically expressed in pre-monocytes. This animal model is a very good one for tracing the development of monocyte towards macrophages in bone marrow.
There are two development pathways of MNCs in BM:
The two pathways were discovered by researchers that using the mice model above.
The CRISPR/Cas9 technology is hot in all areas of bio-med study. It’s useful and easy to handle in modifying all kinds of genes of varied cells. However, the off-target effects of CRISPR/Cas9 sgRNA are unavoidable, thus leading to potential risks of gene modified cells to be used in future in vitro or in vivo study, especially when the cells are going to be used in clinical research.
To detect the off-target sites of CRISPR/Cas9 in gene modified cells is vital. Usually, we first obtain the DNA sequence of off-target sites (OTSs) by input our sgRNA sequence into the website “Cas-OFFinder”, and then we detect the off-targets efficiency of genome DNA in CRISPR/Cas9 excised cells at a certain time post plasmid transduction. If there’re lots of OTSs or the OTSs are in key function genes, we need to think about redesign sgRNA or reschedule the study.
The procedure to detect off-targets when the gene modified cell lines are already established:
- “Cas-OFFinder” get the list of all off-target sites within 3 mismatches and without bulge
- “UCSC_human BLAT search” get exact genomic location info of all OTSs
- “UCSC_human BLAT search” get about ~600bp sequence that carrying each OTS (the flanking sequences of OTS) to be PCR template
- “NCBI_PrimerBlast” design PCR primers for each OTS
- extract DNA of cell lines
- PCR to amplify the DNA sequences that carrying OTS
- PCR products purifying
- Send for Sanger sequencing of purified DNA sequences to detect mutations
- Mutation exist means off-target positive
- To further detect the OT efficiency (ratio of each analyzed OTS): transfect the parental cells of gene modified cell lines again using the same method that established the cell line/ get 2dpt pooled cells to extract genome DNA and send for deep sequencing for each OTS.
Those above are my way to detect off-targets, please leave your comments down below if I made mistakes or you have other suggestions and opinions to share with me!