sdf1-α (cxcl12)  (PeproTech)

Journal: bioRxiv

Article Title: Optimised CRISPR-Cas9 mediated single molecule imaging for accurate quantification through endogenous expression

doi: 10.1101/482596

Figure Lengend Snippet: CRISPR-Cas9 mediated knock-in at the CXCR4 C-terminus results in endogenously labelled protein which localises to the membrane and endosomes. (A) Staining of CXCR4 with KO validated and Fusin antibodies results in poor labelling of the receptor protein, while mEGFP and HaloTag knock-in cells demonstrate a membrane specific localisation. Interestingly mEos 4b CO clones demonstrate fluorescent aggregates, suggesting the fluorophore is cleaved. (B) mEGFP and HaloTag knock-in samples provide clonal populations which are evenly labelled and offer large samples for imaging experiments. (C) Measurement of total CXCR4 expression in wild type versus CRISPR edited cells through qRT-PCR. Compared to wild type, mEGFP knock-ins demonstrate an approximately 20-fold increase in expression (* p = 0.0384). Conversely, HaloTag knock-ins demonstrate a 50% reduction in expression (* p = 0.0301), while mEos 4b CO knock-in shows no change. (D) When RT PCR is performed using tag specific primers, the significant increase in mEGFP expression is likely due to the stability of mEGFP tagged CXCR4 RNA, suggesting that RNA stability is likely a factor in mediating the tag specific down-regulation observed. (E) CXCL12-mediated g protein activation measured by observing G protein dissociation/conformational changes by BRET in knock-in or wildtype cells transfected with cDNA encoding G α i1/Nluc and Venus/G γ 2. Results are expressed as % maximal CXCL12 response observed in wildtype cells andshow a 3 fold increase in response in mEGFP knock-in clones compared to wild type, with a 50% reduction in response in HaloTag knock-in, and no variation in mEos 4b CO clones. (F) Potency of CXCL12-mediated g protein activation in cells expressing wildtype or gene-edited CXCR4.( n = 3 (S.D) for qRT-PCR data, compared by One-Way ANOVA with multiple comparisons. n = 5 for functional data.)

Article Snippet: For CXCL12 treated samples 100ng (10 μ M) recombinant human CXCL12 (SDF1 α , Thermo) was added for 4 minutes prior to fixation.

Techniques: CRISPR, Knock-In, Staining, Clone Assay, Imaging, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Transfection, Functional Assay

Journal: bioRxiv

Article Title: Optimised CRISPR-Cas9 mediated single molecule imaging for accurate quantification through endogenous expression

doi: 10.1101/482596

Figure Lengend Snippet: CRISPR Knock-In CXCR4 clones allow for accurate single molecule cluster analysis compared to transfected cells. (A) Single molecule imaging of HaloTag CXCR4 Knock-Ins allows for effective single molecule localisation microscopy through labelling with Janelia Fluor 549, and subsequent cluster analysis using persistence based clustering. (B) CXCR4 knock-in and Hek293T transfected with a CXCR4-HaloTag were subject to single molecule imaging and cluster analysis after treatment with CXCL12 (10 μ M). CRISPR knock-ins demonstrate a significant increase in median cluster area on ligand treatment (* p = 0.0272), while transfected cells show no significant change.(C) Histograms showing percentage of clusters across a range of areas show that while a substantial shift towards larger cluster sizes is evident in CRISPR knock-ins, this is reduced in transfected cells. (D) The percentage of clusters below 50nm in radius is significantly reduced in CRISPR cells (* p = 0.0384) upon treatment with CXCL12 (upper panel), with a parallel increase in the percentage of clusters of larger sizes consistent with endocytic trafficking observed (* p = 0.0133) (lower panel). Conversely, no significant change in either collection of cluster sizes is evident in transfected cells. ( n = 3, S.E.M). paired t-test.)

Article Snippet: For CXCL12 treated samples 100ng (10 μ M) recombinant human CXCL12 (SDF1 α , Thermo) was added for 4 minutes prior to fixation.

Techniques: CRISPR, Knock-In, Clone Assay, Transfection, Imaging, Microscopy