Oct 23, 2024
Cell line construction and analysis of agDD-GFP protein aggregates
Forked from a private protocol
- Harper JW1,2,
- Kelsey Hickey1,2
- 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Harper JW, Kelsey Hickey 2024. Cell line construction and analysis of agDD-GFP protein aggregates. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyr7kl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2024
Last Modified: October 23, 2024
Protocol Integer ID: 110232
Keywords: ASAPCRN, protein aggregates, proteasome, flow cytometry
Funders Acknowledgement:
ASAP
Grant ID: 000282
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Abstract
Here, we describe protocols for analysis of the inducible protein aggregate agDD-GFP in HeLa-dCas9 and HEK293T cells under a variety of conditions and in various genetic backgrounds. agDD-GFP is stabilized by a small molecule (shield-1, S1) and washout of S1 leads to agDD-GFP aggregation in minutes. By monitoring aggregates after S1 washout by flow cytometry or microscopy, it is possible to observe proteasome-dependent turnover of agDD-GFP proteins. Methods (flow cytometry) and cell lines to observe this turnover are described in this protocol.
Materials
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Chemicals, peptides, and recombinant proteins | |||
| Puromycin | Sigma-Aldrich | P9620 | |
| G418 (Geneticin) | Invivogen | ant-gn-2 | |
| Dulbecco’s MEM (DMEM), high glucose, pyruvate | GIBCO / Invitrogen | 11995 | |
| fetal calf serum | |||
| NLS-Cas9 protein | Berkeley QB3 MacroLab | https://qb3.berkeley.edu/facility/qb3-macrolab/projects/#cas9-nls-purified-protein | |
| S1 | MedChem Express | # HY-112210 | |
| Experimental models: Cell lines | |||
| HeLa-dCas9 cells | Ref 2 | Ref 2 | |
| HEK293T | ATCC | RRID: CVCL_0063 | |
| Recombinant DNA | |||
| Plasmid expressing agDD-GFP | Ref 1 | Addgene #78289 | |
| pCRISPRia-v2 | Ref2 | Addgene #84832 | |
| pU6-sgRNA EF1a-puro-t2a-mCherry | unpublished | Addgene #217306 | |
| Oligonucleotide | |||
| GAPDH: Forward-acgggaagcttgtcatcaat | IDT | qPRC primer | |
| GAPDH: Reverse-catcgccccacttgatttt | IDT | qPRC primer | |
| NRF1: Forward-ggaacagcagtggcaagatctc | IDT | qPRC primer | |
| NRF1: Reverse-gcaaggctgtagttggtgctca | IDT | qPRC primer | |
| DDI2: Forward-tgctgaaggaacgcaatccacc | IDT | qPRC primer | |
| DDI2: Forward-tgctgaaggaacgcaatccacc, Reverse-cagacgaatcctttcttgctctc | IDT | qPRC primer | |
| UBE3A: Forward-cgaagaatcactgttctctacagc | IDT | qPRC primer | |
| UBE3A: Reverse-ggattttccatagcgatcatct | IDT | qPRC primer | |
| UBE3B: Forward-aagctctgcggaactgtcat | IDT | qPRC primer | |
| UBE3B: Reverse-cggtgttcgctttcagaac | IDT | qPRC primer | |
| UBE3C: Forward-ctgccaggatgttcagcttc | IDT | qPRC primer | |
| UBE3C: Reverse-tttcttcctgagtacgatgtaaaaga | IDT | qPRC primer | |
| PSMD4: Forward-ctggctaaacgcctcaagaagg | IDT | qPRC primer | |
| PSMD4: Reverse-cactgtcaccagatgagaaccg | IDT | qPRC primer | |
| PSMB5: Forward-gtgtcccagaagagccaggaat | IDT | qPRC primer | |
| PSMB5: Reverse-tcttcaccgtctgggaggcaat | IDT | qPRC primer | |
| UBE3A-GGGCCGCGGCGCAAGACGGG | IDT | Protospacer sequence | |
| UBE3B-GCCCGGGTCTGGCAGAACTC | IDT | Protospacer sequence | |
| UBE3C-GCACAGCTCGGGCCGCTGCA | IDT | Protospacer sequence | |
| NRF1-GAAGCTCCGGCGCCGAGAGTG | IDT | Protospacer sequence | |
| DDI2-GACTCACTGAGCGTGTGTGAG | IDT | Protospacer sequence | |
| Forward gRNA primer sequence for UBE3C knockout: TAATACGACTCACTATAGGAGCTACGAAGACGATGTGG | IDT | Primer for tracr gRNA synthesis | |
| Reverse gRNA primer sequence for UBE3C knockout: TTCTAGCTCTAAAACCCACATCGTCTTCGTAGCTC | IDT | Primer for tracr gRNA synthesis | |
| Forward gRNA primer sequence for knockin of RPN13K21R: TAATACGACTCACTATAGGGGCGCCTCCAACAAGTACT | IDT | Primer for tracr gRNA synthesis | |
| Reverse gRNA primer sequence for knockin of RPN13K21R: TTCTAGCTCTAAAACAGTACTTGTTGGAGGCGCCC | IDT | Primer for tracr gRNA synthesis | |
| Forward gRNA primer sequence for knockin of RPN13K34R: TAATACGACTCACTATAGGAGTCACGGTGGTCCCCTTC | IDT | Primer for tracr gRNA synthesis | |
| Reverse gRNA primer sequence for knockin of RPN13K34R: TTCTAGCTCTAAAACGAAGGGGACCACCGTGACTC | IDT | Primer for tracr gRNA synthesis | |
| ultramer for homology-directed knockin of RPN13K21R;K34R: CTCTTTCCAAGCCTGGTGCCAGGCTCTCGGGGCGCCTCCAACAGGTACTTGGTG GAGTTTCGGGCGGGAAAGATGTCCCTGAGGGGGACCACCGTGACTCCGGATAAGCGGAAAGGGCTGGTGTACATTCAGCAGACGGAC | IDT | gRNA sequence | |
| UBE3C MiSeq Forward-TAATACGACTCACTATAGGAGCTACGAAGACGATGTGG | IDT | MiSeq | |
| UBE3C MiSeq Reverse-TTCTAGCTCTAAAACCCACATCGTCTTCGTAGCTC | IDT | MiSeq | |
| RPN13 MiSeq Forward-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTTCAGGATGACGACCTCAG | IDT | MiSeq | |
| RPN13 MiSeq Reverse-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGTACACCAGCCCTTTC | IDT | MiSeq |
Cell line maintenance
Cell line maintenance
Maintain HeLa-dCas9 and HEK293T cells in complete DMEM (Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1% penicillin-streptomycin).
agDD-GFP (Addgene Plasmid #78289) is integrated into HeLa-dcas9 and HEK293T cells using the piggy-bac transposase, grown for 10 days, and then sorted for GFP positive cells. After integration maintain cells in complete DMEM that contains shield-1 ligand at a final concentration of 1 μm.
Expression of CRISPRi targeting vectors in HeLa-dCas9 cells
Expression of CRISPRi targeting vectors in HeLa-dCas9 cells
Prepare vectors for expression of gRNAs. Single sgRNA expression vectors were individually cloned by annealing complementary synthetic oligonucleotide pairs (Integrated DNA Technologies) for each sgRNA with flanking BstXI and BlpI restriction sites and ligating the resulting double-stranded segment into either BstXI/BlpI-digested pCRISPRi-v2 (Addgene #84832) [marked with a puromycin resistance cassette and BFP as described (2)] or BstXI/BlpI-digested pU6-sgRNA EF1a-puro-t2a-mCherry Addgene #217306) (marked with a puromycin resistance cassette and RFP). Oligonucleotide sequences are provided in the materials table.
The resulting sgRNA expression vectors are individually packaged into lentivirus.
These sgRNA vectors were transduced into HeLadCas9 +agDD-GFP cells at MOI < 1 (20 – 40% infected cells).
Select cells with puromycin (1 microgram/ml) for 48 hours, then allow additional 48 hours of recovery.
Allow 5-8 days for knockdown [measured by qPCR] before assaying phenotypes.
Gene editing of UBE3C knock-out and RPN13 knock-in in HEK293T cells
Gene editing of UBE3C knock-out and RPN13 knock-in in HEK293T cells
Forward and reverse oligonucleotides that contain CRISPR target sequences for UBE3C or RPN13 were ordered from IDT, which were used to generate gRNA by Precision gRNA Synthesis kit (Thermo Fischer, A29377). Briefly, the gene-specific primers were coupled with Phusion DNA polymerase, Tracr Fragment and T7 primer mix to generate the full-length gRNA DNA template, followed by in vitro transcription and gRNA purification.
Gene-specific primers for UBE3C and RPN13 are as follows:
Forward gRNA primer sequence for UBE3C knockout: TAATACGACTCACTATAGGAGCTACGAAGACGATGTGG
Reverse gRNA primer sequence for UBE3C knockout: TTCTAGCTCTAAAACCCACATCGTCTTCGTAGCTC
Forward gRNA primer sequence for knockin of RPN13K21R: TAATACGACTCACTATAGGGGCGCCTCCAACAAGTACT
Reverse gRNA primer sequence for knockin of RPN13K21R: TTCTAGCTCTAAAACAGTACTTGTTGGAGGCGCCC
Forward gRNA primer sequence for knockin of RPN13K34R: TAATACGACTCACTATAGGAGTCACGGTGGTCCCCTTC
Reverse gRNA primer sequence for knockin of RPN13K34R: TTCTAGCTCTAAAACGAAGGGGACCACCGTGACTC
Electroporation was performed using the Neon Electroporation system (Invitrogen, MPK5000) and the Neon Transfection Kit (Invitrogen, MPK1096B). 1.2 micrograms of gRNA and 6 micrograms of Cas9 protein were mixed in 10ul buffer R and were incubated for 10 minutes to allow the RNP complex to form. Cells were detached and resuspended in buffer R (a component of the Neon transfection kit) at a concentration of 2x10^5 cells per 5ul. 10ul of cell suspension was mixed with RNP complex, which is enough for 2 transfections. To make RPN knock-in cells, 78 micromol of the ultramer was added right before adding cells to the mixture
Ultramer for homology-directed knockin of RPN13 K21R and K34R: CTCTTTCCAAGCCTGGTGCCAGGCTCTCGGGGCGCCTCCAACAGGTACTTGGTG GAGTTTCGGGCGGGAAAGATGTCCCTGAGGGGGACCACCGTGACTCCGGATAAGCGGAAAGGGCTGGTGTACATTCAGCAGACGGAC
To create the K21R:K34R double knock-in cells, sequential targeting first with the K34R guide and then with the K21R guide was performed.
After 48 hours of cell recovery, cells were single cell sorted into 96-well plates by FACS to isolate single
clones. After 1-2 weeks, single cells that grew to colonies were split into two sets: one for Illumina MiSeq
sequencing analysis, the other for western blotting.
Clones are identified as knockouts by Illumina MiSeq sequencing and further confirmed by western blotting.
agDD-GFP analysis by flow cytometry
agDD-GFP analysis by flow cytometry
Cells were maintained in S1 to maintain agDD-GFP in a soluble folded form (1 μm S1).
To initiate aggregation, S1-containing mediate was aspirated from the cells, and
conditioned media containing 5 μm FKBP12F36V (5) was added to cells.
After aggregation timecourse, cells were treated with trypsin and quenched with Phenol red free-DMEM. Cells were filtered through a cell strainer cap tube and analyzed on an Attune NxT (Thermo Fisher). At least 10,000 single, healthy cells were analyzed for each condition. Median fold changes of GFP were calculated and plotted using FlowJo Software.
Immunoblotting of NRF1
Immunoblotting of NRF1
Cells were cultured in the presence of the corresponding stress to 60-80% confluency in 6-well plates, 10 cm or 15 cm dishes. After removing the media, the cells were washed with DPBS three times. Cells were resuspended in lysis buffer (50 mM TRIS 7.5, 150 mM NaCl, 0.5% NP-40, containing mammalian protease inhibitor cocktail (Sigma), Phos-STOP, and 20 unit/ml Benzonase (Millipore)). Cell lysates were cleared by centrifugation (15000 rpm, 10 min at 4 oC).
The concentration of the supernatant was measured by BCA assay. 20ug of cell lysate [treated with endoH or untreated] was denatured by the addition of LDS sample buffer supplemented with 100 mM DTT, followed by boiling at 95oC for 5 minutes and loaded onto the 8% or 4-12% NuPAGE Bis-Tris gel (Thermo Fisher Scientific), followed by SDS-PAGE with MOPS SDS running buffer (Thermo Fisher Scientific).
The proteins were electro-transferred to nitrocellulose membranes and then the total protein was stained using Ponceau (Thermo Fisher Scientific). The membrane was then blocked with LI-COR blocking buffer at room temperature for 1h. Then membranes were incubated with the indicated primary antibodies (4oC, overnight), washed three times with TBST (total 30 min), and further incubated either with fluorescent IRDye 680RD Goat anti-Mouse IgG H+L, or IRDye 800CW Goat anti-Rabbit IgG H+L secondary antibody at (1:10,000) at room temperature for 1h.
After thorough wash with TBST for 30 min, near infrared signal was detected using OdysseyCLx imager and quantified using ImageStudioLite (LI-COR).
Over expression of NRF1
Over expression of NRF1
NRF1 (Addgene #181917) and NRF1 18ND (Addgene #181918) (10) overexpression
plasmids from were packaged into lentivirus followed by transduction of HeLadCas9
agDD-GFPHIGH cells.
48 hours after infection, cells were selected with puromycin (1 microgram/ml) for 48 hours, recovered for an additional 48 hours.
Aggregation is started for various lengths of time and measured by flow cytometry in control and over expression cell lines. Ectopic expression of NRF1 or the 18ND mutant is verified by immunoblotting of cell extracts.
Analysis of gene knockdown efficiency and NRF1 pathway transcription by qPCR
Analysis of gene knockdown efficiency and NRF1 pathway transcription by qPCR
Harvest cells that have been selected with puromycin and recovered [5-8 days post infection] for knockdown analysis. For NRF1 pathways transcription experiments, plate selected cells and perform aggregation timecourse. qPCR primers for GAPDH, UBE3A, UBE3B, UBE3C, NRF1, DDI2, PSMD4, and PSMB5 are provided in the materials table.
Total RNA was isolated from ~1 million frozen cell samples using Direct-zol RNA MiniPrep kit as described by the manufacturer.
Reverse-transcription was carried using SSIII Reverse-transcriptase (Thermo Fisher Scientific) with oligodT primer (Thermo Fisher Scientific, SO124) in the presence of RNaseIN Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).
Quantitative PCR (qPCR) (in technical triplicates) was performed with Kappa Sybr Fast qPCR 2x Mix with low ROX (Roche), according to the manufacturer’s instructions on Thermo pro quant 7.
Protocol references
1. Y. Miyazaki et al., A method to rapidly create protein aggregates in living cells. Nat Commun 7, 11689 (2016).
2. L. A. Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451 (2013).