Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9

使用多色Lenti-CRISPR-Cas9高效生成多基因敲除细胞系和患者源性异种移植物

LB Lena Behrmann *
Scott McComb Scott McComb *
JA Júlia Aguadé-Gorgorió
YH Yun Huang
MH Mario Hermann
PP Pawel Pelczar
AA Adriano Aguzzi
JB Jean-Pierre Bourquin
Beat C. Bornhauser Beat C. Bornhauser

 (*contributed equally to this work) 发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2222 浏览次数: 20038

评审: Gal HaimovichAnnis Elizabeth RichardsonAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Science Translational Medicine, featuring study using the protocol.

May 2016

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

边缘间充质基质细胞的分离和离体扩增

边缘间充质基质细胞的分离和离体扩增

Naresh Polisetti [...] Günther Schlunck

2022年07月20日 2158 阅读

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

Chloe A. L. Thompson-Peach [...] Daniel Thomas

2023年01月20日 1486 阅读

表达绿色荧光蛋白的报告小鼠胚胎黑素细胞的分离、纯化和培养

表达绿色荧光蛋白的报告小鼠胚胎黑素细胞的分离、纯化和培养

Melissa Crawford [...] Lina Dagnino

2023年09月05日 1274 阅读

Abstract

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

Keywords: CRISPR (CRISPR) search Cas9 (Cas9) search Multiple gene knockout (多基因敲除) search Lentivirus (慢病毒) search Primary human leukemia (原发性人白血病) search Xenograft (异种移植物) search sgRNA design (sgRNA设计) search

Background

Starting with curious initial observations of genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) within bacterial genomes (Ishino et al., 1987; Mojica et al., 2000) and subsequent gene editing in mammalian cells (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas9 has become the cutting edge option for inexpensive and efficient gene editing. With successful application in cellular systems ranging from tobacco plant cells to zebrafish and primary human cells (Hsu et al., 2014), CRISPR-Cas9 can be directed by design of a short 20 nucleotide RNA sequence to create targeted DNA double strand breaks (DSB) within large genomes (Park et al., 2016). After DSBs occur, cells can initiate repair either through high fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ), often leading to small insertion and deletion (indel) mutations resulting in gene knockout (Gaj et al., 2013; Bétermier et al., 2014) (Figure 1).


Figure 1. Principle of genome editing by CRISPR Cas9. The principle of a gene knockout by CRISPR-Cas9 is shown exemplarily for the RIP1 sequence. A. Single guided RNA (sgRNA) consists of the target sequence specific crRNA (CRISPR RNA) and the constant tracrRNA (trans-activating crRNA) (Jinek et al., 2012). crRNA is binding to the genomic DNA adjacent to the PAM motif and tracrRNA guides the Cas9 enzyme to the locus. B. Cas9 mediated DNA double strand breaks (DSB) activate non-homologous end joining (NHEJ). C. Imprecise DSB repair leads to gain or loss of nucleotides (indels) with a two-thirds chance of causing frameshift mutations that may result in the generation of premature stop codons.

A number of different strategies have emerged to deliver Cas9 protein and targeting RNA into cells, including electroporation or transfection of Cas9/sgRNA ribonucleoprotein complexes, mRNA, plasmid or lentiviral vectors carrying sgRNA and Cas9 payloads (Sander and Joung, 2014; Shalem et al., 2014). Previously these LentiCRISPR plasmids carried a resistance gene to allow selection of cells with constitutive expression of the machinery necessary for CRISPR-Cas9-directed gene disruption.

As shown in our recent publication (McComb et al., 2016), we have adapted the LentiCRISPR protocol for directed disruption of several genes simultaneously in cell lines and primary leukemia cells based on selection by fluorescence combined with fluorescence-activated cell sorting (FACS). By swapping the puromycin resistance gene for fluorescent protein markers (EGFP, mCherry, tagBFP, or RFP657), up to four genes can be simultaneously targeted for CRISPR-Cas9-mediated gene disruption. Fluorescence-activated cell sorting enables isolation of cell lines or primary human cells bearing sgRNAs targeting one to four genes in one single experimental step. Our multi-color LentiCRISPR technique thus allows the simultaneous generation of knockout cells bearing anywhere between one and four gene knockouts, allowing rapid testing of gene-gene interaction within a set of genes of interest. The backbone vectors with the four different fluorescence markers as well as the herein described target constructs including cloning information have been deposited at Addgene.

Here we provide a complete step-by-step guide protocol to generate single and multi-gene knockout cells by multicolor LentiCRISPR (see Figure 2 for schematic overview).


Figure 2. Schematic overview of the procedure to generate multicolor LentiCRISPR knockout cell lines and patient derived xenografts

Development of the protocol
To study the regulation of cell death in leukemia, we developed multi-color LentiCRISPR as a tool to target proteins essential for two divergent pathways of programmed cell death, apoptosis and necroptosis (McComb et al., 2016). Efficient deletion of the respective targets, like RIP1, RIP3, MLKL, FADD and CASP8, was demonstrated by Western blot analysis of protein in targeted compartments (see Data analysis). Through simultaneous gene disruption, we showed that it is necessary to inactivate both apoptosis and necroptosis within leukemic cell lines and patient-derived xenografts in order to render cells resistant to SMAC mimetics, a specific class of chemotherapeutic compounds targeting the inhibitor of apoptosis proteins, IAPs. These data provide convincing evidence that both apoptosis and necroptosis can independently kill leukemia cells in vivo, and are a strong proof of concept for the multicolor LentiCRISPR technique as a means to investigate gene redundancy.

Experimental design
sgRNA design and preparation. We first describe a fast and easy way to design and clone sgRNAs for any target gene with a single pot reaction for restriction and ligation (Figure 3). We have had good success utilizing the CRISPR design online tool (http://cripr.mit.edu) from the Massachusetts Institute of Technology, developed by the lab of Feng Zhang to predict binding sites for Cas9 with minimal risk of off-target activity (Hsu et al., 2013). Alternative sgRNA prediction software (such as http://crispor.tefor.net/) can also be used to provide in silico prediction of sgRNA-specific cleavage activity based on a number of different algorithms. However, we still recommend the design of three sgRNAs per target gene and assessing their gene knockout activity in cell lines before moving on to more challenging applications. Strategies targeting only the 5’ exon of candidate genes might induce in-frame mutations that can retain the full protein functionality. A recent publication showed that targeting particular exonic regions with key functional protein domains increases the chance of null mutations without a full protein knockout (Shi et al., 2015). For this reason, we suggest spreading sgRNA candidates among different exons to increase the probability of achieving a potent gene deletion.


Figure 3. Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. Primer design is shown exemplarily for RIP1. A. From the genomic sequence the target locus for CRISPR editing was chosen and screened by http://crispr.mit.edu for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp3I restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.

Lentiviral production and infection. This protocol makes use of multicolor LentiCRISPR plasmids cloned from one-vector LentiCRISPR system developed by the Zhang lab (Shalem et al., 2014). Protocol conditions have been optimized for the transduction of acute lymphoblastic leukemia (ALL) cell lines and patient derived xenograft ALL cells from previously published protocols (Tiscornia et al., 2006; Kutner et al., 2009; Weber et al., 2012). Optimized conditions are recommended for every cell line or patient-derived sample. Here, we describe the production of lentiviral particles with the VSV-G envelope, because it is known for its high titers and broad tropism. Depending on the target cells the pseudotyping can be exchanged. For murine applications, the exchange to a mouse ecotropic envelope protein enhances safety and enables the use of the lentiviral vectors under biosafety level 1 conditions.

Analysis of knockout efficiency
After purification of cells transduced with Cas9/sgRNA targeted against a gene of interest, it is straightforward to confirm knockout at protein level (measurement via flow cytometry, ELISA or Western blot). Thus it is essential that a specific antibody for your protein of interest is available (see Figure 4 for further discussion and considerations for single cell cloning). Regardless of the viral transduction efficiency and the gene-editing efficacy of the selected sgRNA/CRISPR-Cas9 construct, DNA mutations do not invariantly lead to a loss of protein. Based on the triplet coding sequences the ratio of a frameshift after NHEJ is 2:1 resulting in possible indels without frameshift. Depending on the location of indel formation, this may also lead to unpredictable effects on mRNA or protein stability. Thus, we do not recommend knockout confirmation at DNA or RNA level by sequencing or SURVEYOR nuclease assay since this does not confirm the loss of the protein expression and function. Lesions can lead to a loss of amino acids and a change in protein functionality, but can also result in a slightly impaired protein with a near wild type function. Depending on the target gene a functional assay can be performed (i.e., enzyme activity) or cellular localization can be visualized (i.e., for nuclear receptors) but we recommend performing Western blots or ELISA to quantify protein level.

Limitations of the technique
Lentiviral vector efficiently delivers the CRISPR machinery to a wide range of cell lines and primary cells. However, as with any viral approach, there is a risk of insertional mutagenesis, although this can be minimized by transducing cells at a low multiplicity of infection (MOI) to limit the number of integration events. Constitutive expression of Cas9/sgRNA may also lead to accumulation of mutations at off-target sites, stressing the need for good sgRNA design to limit off-target binding.

Efficiency of knockouts using LentiCRISPR can vary significantly depending on the specific genes targeted, especially for targets that confer a selective advantage/disadvantage to knockout cells. It might thus be a benefit to utilize an inducible CRISPR plasmid for such applications. In this case, Cas9 or sgRNA expression would be controllable in vitro and in vivo by administration of i.e., doxycycline.
Our strategy also depends on the reliability of the detection of reporter fluorescence. There is some evidence for silencing of expression over time for fluorescent proteins of the GFP family when expressed from lentiviral vectors. The populations should therefore be continuously monitored for expression. While lentiviral delivery has been proven to be very efficient in many different cellular systems, transduction efficiency heavily depends on the size of the delivered plasmids (Canté-Barrett et al., 2016). The large size of the LentiCRISPR vector is known to lead to low viral titers, nonetheless, viral concentration and other optimizations in the protocol below have allowed us to successfully apply these vectors in hard to transduce leukemic cell lines and primary cells.


Figure 4. Considerations for single cell cloning. We recommend single cell cloning if a clonal cell population with a constant genetic background is desired for long term experimentation. Here we present a Western blot confirmation of the knockout of RIP1 in either (A) double sorted or (B) single cell cloned Jurkat cells. Irrespective the purity of the sorted cell population, a minor RIP1 signal remains in the double sorted population, whereas in single cell clones a pure knockout can be achieved. Proteins were detected with mouse anti-RIP1 (1:1,000) and goat anti-mouse-HRP (1:5,000).

Materials and Reagents

  1. Filtered sterile pipette tips
    10 µl (STARLAB INTERNATION, catalog number: S1121-3810 )
    200 µl (STARLAB INTERNATION, catalog number: S1120-8810 )
    1,000 µl (STARLAB INTERNATION, catalog number: S1126-7810 )
  2. TC plate
    6 well (SARSTEDT, catalog number: 83.3920 )
    24 well (SARSTEDT, catalog number: 83.3922 )
    96 well R (SARSTEDT, catalog number: 83.3925 )
  3. Filters: Filtropur S
    0.22 µm (SARSTEDT, catalog number: 83.1826.102 )
    0.45 µm (SARSTEDT, catalog number: 83.1826 )
  4. Serological pipettes
    5 ml (SARSTEDT, catalog number: 86.1253.001 )
    10 ml (SARSTEDT, catalog number: 86.1254.001 )
    25 ml (SARSTEDT, catalog number: 86.1685.001 )
  5. Amicon Ultra-15 centrifugal filter units (EMD Millipore, catalog number: UFC900308 )
  6. Tube
    15 ml (SARSTEDT, catalog number: 62.554.502 )
    50 ml (SARSTEDT, catalog number: 62.547.254 )
    13 ml for bacteria culture (SARSTEDT, catalog number: 62.515.006 )
  7. SafeSeal tubes 1.5 ml (SARSTEDT, catalog number: 72.706 )
  8. Nitrocellulose membranes (Trans-Blot Turbo transfer pack) (Bio-Rad Laboratories, catalog number: 170-4159 )
  9. Disposable syringe 10 ml (CODAN, catalog number: 62.6616 )
  10. Injectomat syringe 50 ml (Fresenius Kabi, catalog number: 9000711 )
  11. Petri dishes (92 x 16 mm) for LB plates (SARSTEDT, catalog number: 82.1472.001 )
  12. Round-bottom tube, 5 ml (Corning, Falcon®, catalog number: 352052 )
  13. Round-bottom tube with cell strainer cap, 5 ml (Corning, Falcon®, catalog number: 352235 )
  14. TC flask T175, vent. cap (SARSTEDT, catalog number: 83.3912.002 )
  15. Microvette 100 LH, Lithium-Heparin (SARSTEDT, catalog number: 20.1282 )
  16. NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ) mice (RRID:IMSR_ARC:NSG), age ~4-6 weeks
    Caution: Institutional animal care guidelines must be followed. Here the animal experiments were approved by the veterinary office and the ethics commission of the Canton of Zurich, Switzerland.
  17. HEK293T cells (ATCC, catalog number: CRL-3216 )
    Note: Cells should be growing actively in culture with DMEM medium with 10% (v/v) FBS. Cells should be maintained between 10-90% confluence, splitting at least twice per week.
  18. Target cells
    Note: Cells should be growing in healthy culture with appropriate medium. For most ALL cell lines RPMI with 10% (v/v) FBS is suitable. Primary cells should be thawed a day before transduction.
    Caution: Primary human cells have to be considered potentially hazardous. Primary derived xenografts were obtained from human ALL samples recovered from cryopreserved bone marrow aspirates. Here patients were enrolled in the ALL-BFM 2000 and ALL-BFM 2009 studies. Informed consent was given in accordance with the Declaration of Helsinki and approval was granted by the Ethics Commission of the Kanton Zurich.
  19. One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404010 or equivalent)
  20. CRISPR target plasmid with BsmBI (Esp3I) insert site, e.g.,
    pLentiCRISPR-EGFP (Addgene, catalog number: 75159 )
    RFP657 (Addgene, catalog number: 75162 )
    BFP (Addgene, catalog number: 75160 )
    mCherry (Addgene, catalog number: 75161 )
  21. psPAX2 plasmid (Addgene, catalog number: 12260 )
  22. p.CMV.VSV.G plasmid (Addgene, catalog number: 8454 )
    Caution: It may cause an allergic skin reaction or asthma symptoms.
  23. Oligonucleotides 100 µM, standard synthesis, desalted purification (for example sequences see Table 1)

    Table 1. Sequences of oligonucleotides for pLentiCRISPR cloning


  24. Tango buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: BY5 )
  25. T4 DNA ligase (New England Biolabs, catalog number: M0202M ) make aliquots of T4 DNA ligase buffer and use only freshly thawed aliquots for reaction
  26. Esp3I restriction enzyme (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: ER0451 )
    Note: NOT NEB BsmBI, BsmBI requires 55 °C for efficient cleavage – not compatible with this protocol.
  27. SOC medium (Sigma-Aldrich, catalog number: S1797 or equivalent)
  28. Ampicillin (Sigma-Aldrich, catalog number: A9518 or equivalent)
  29. RED Taq Ready Mix PCR Reaction mix (Sigma-Aldrich, catalog number: R2523 or equivalent)
  30. Mini- and Midi-plasmid Preparation Kit (QIAGEN, catalog number: 27106 or equivalent)
  31. Tris-acetate-EDTA buffer (TAE, 25x) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9870 or equivalent)
  32. Polyethylenimin (PEI transfection reagent) (Sigma-Aldrich, catalog number: 408727 )
    Note: You must test your preparation of PEI prior to use to establish the concentration, which is necessary to get the best transduction efficiency. We generally use ~20 µg/ml final concentration.
    Caution: It is toxic if swallowed.
  33. ProFection Mammalian Transfection System (Promega, catalog number: E1200 )
  34. Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 or equivalent)
    Caution: It is harmful if swallowed.
  35. Calcium chloride (CaCl2)
  36. HBS
  37. Polybrene (hexadimethrine bromide) (Sigma-Aldrich, catalog number: H9268 ) stock solution 8 mg/ml in ddH2O
    Caution: It is harmful if swallowed.
  38. Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D1408 or equivalent)
  39. Trypsin (0.05% in PBS) with EDTA (BioConcept, catalog number: 5-51F00-I or equivalent)
  40. Formalin (4% formaldehyde) (Formafix, catalog number: 01-1010 )
  41. Polyethylene glycol (PEG 6000) (Sigma-Aldrich, catalog number: 81253 )
  42. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 or equivalent)
  43. RetroNectin Recombinant Human Fibronectin Fragment (Takara Bio, Clontech, catalog number: T100B )
    Note: Prepare Retronectin according to manufacturer’s protocol, aliquot and store at -20 °C. Retronectin can be reused up to 4 times without quality decrease.
  44. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 or equivalent)
  45. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, catalog number: 14170088 or equivalent)
  46. HEPES, 1 M (Thermo Fisher Scientific, catalog number: 15630056 or equivalent)
    Caution: It can cause skin irritation and serious eye irritation.
  47. Flow cytometry antibody (PE-Cy7 anti-human CD19) (BioLegend, catalog number: 302216 , RRID:AB_314246)
  48. NuPage MES SDS 20x running buffer (Thermo Fisher Scientific, NovexTM, catalog number: NP0002-02 ) dilute 1:20 with ddH2O for final concentration
  49. Pageruler Prestained Protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26616 or equivalent)
  50. Precast gels (Criterion XT 4-12% Bis-Tris 1.0 mm gels)
    18 well (Bio-Rad Laboratories, catalog number: 3450124 )
    26 well (Bio-Rad Laboratories, catalog number: 3450125 )
  51. Nonfat-dried milk (Sigma-Aldrich, catalog number: M7409 or equivalent)
    Note: Prepare 5% (w/v) working solution in 1x TBS-T. Prepare fresh and store at 4 °C for short term.
  52. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 or equivalent)
    Caution: It is fatal if swallowed or in contact with skin. May cause damage to organs if swallowed. Very toxic to aquatic life.
  53. Secondary HRP-conjugated antibody
  54. Western blot primary antibodies
    Mouse anti-RIP1 (BD, BD Biosciences, catalog number: 551042 , RRID:AB_394015)
    Rabbit anti-FADD (Cell Signaling Technology, catalog number: 2782 , RRID:AB_2100484)
    Mouse anti-Tubulin (Sigma-Aldrich, catalog number: T9026 , RRID:AB_477593)
    Rat anti-MLKL (EMD Millipore, catalog number: MABC604 )
    Mouse anti-CASP8 (Cell Signaling Technology, catalog number: 9746 , RRID:AB_2068482)
    Rabbit anti-RIP3 (Abnova, catalog number: PAB0287 , RRID:AB_1019004)
  55. Western blot secondary antibodies conjugated to horseradish peroxidase
    Goat anti-mouse (Cell Signaling Technology, catalog number: 7076 , RRID:AB_330924)
    Goat anti-rabbit (Cell Signaling Technology, catalog number: 7074 , RRID:AB_10697506)
    Goat anti-rat (Cell Signaling Technology, catalog number: 7077 , RRID:AB_10694715)
  56. Baytril, Bayer, 0.5 ml in 250 ml autoclaved drinking water (stock 2.5%)
  57. Gene Ruler 1 kb Plus DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 or equivalent)
  58. GelRed, 10,000x (Biotium, catalog number: 41003 )
    Note: GelRed is superior to ethidium bromide by having low toxicity and high sensitivity.
  59. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 or equivalent)
    Caution: It is dangerous. Causes severe skin burns and eye damage.
  60. SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 34096 )
  61. Benzonase Nuclease (Sigma-Aldrich)
  62. Bacto tryptone (BD, BactoTM, catalog number: 211705 or equivalent)
  63. Yeast extract (BD, BactoTM, catalog number: 212750 or equivalent)
  64. Agar (BD, BactoTM, catalog number: 214010 or equivalent)
  65. Agarose (Eurogentec, catalog number: EP-0010-05 or equivalent)
  66. DMEM medium (Sigma-Aldrich, catalog number: D5546 or equivalent)
  67. Fetal calf serum (FCS) (Sigma-Aldrich, catalog number: 12133C or equivalent)
  68. L-glutamine, 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 or equivalent)
  69. Sodium pyruvate, 100 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 or equivalent)
  70. RPMI medium (Sigma-Aldrich, catalog number: R8758 or equivalent)
  71. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 or equivalent)
  72. FBS
  73. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 or equivalent)
  74. Trizma HCl (Sigma-Aldrich, catalog number: T5941 or equivalent)
  75. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 or equivalent)
    Caution: It is harmful if swallowed and toxic in contact with skin. Flammable solid. Can cause eye and skin irritation and may cause respiratory irritation.
  76. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 or equivalent)
  77. Glycerol (Sigma-Aldrich, catalog number: G5516 or equivalent)
  78. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 or equivalent)
    Caution: It is toxic if swallowed or if inhaled. Fatal in contact with skin. Causes skin irritations and eye damage.
  79. Ammonium chloride (NH4Cl) (Carl Roth, catalog number: K298.1 or equivalent)
    Caution: It is harmful if swallowed. It causes serious eye irritation.
  80. Potassium chlorate (KClO3) (EMD Millipore, catalog number: 104854 or equivalent)
    Caution: It is dangerous and harmful if swallowed or inhaled. May cause fire, is a strong oxidizer.
  81. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 or equivalent), prepare 0.5 M stock solution with ddH2O and pH 8.0 with NaOH
    Caution: It is harmful if inhaled.
  82. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 or equivalent)
  83. Trizma base (Sigma-Aldrich, catalog number: 93352 or equivalent)
  84. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 or equivalent)
    Caution: It is toxic if inhaled. Causes severe skin burns and eye damage.
  85. Tween 20 (Sigma-Aldrich, catalog number: P9416 or equivalent)
  86. Ponceau S (Sigma-Aldrich, catalog number: P3504 )
  87. Acetic acid (Sigma-Aldrich, catalog number: A6283 or equivalent)
    Caution: It is flammable and causes severe skin burns and eye damage.
  88. LB agar plates Combine (see Recipes)
  89. LB medium Combine (see Recipes)
  90. DMEM complete (see Recipes)
  91. RPMI medium (see Recipes)
  92. Freezing medium (see Recipes)
  93. 3x SDS lysis buffer (see Recipes)
  94. 1x SDS loading buffer (see Recipes)
  95. Red blood lysis buffer (RBC buffer) (see Recipes)
  96. 10x TBS (see Recipes)
  97. 1x TBS-T (see Recipes)
  98. Ponceau S solution (see Recipes)
  99. SDS Glycine stripping buffer (see Recipes)

Equipment

  1. Pipettes (Gilson)
  2. Pipetus pipetting aid (Hirschmann Laborgeräte)
  3. Shaker (Thomas Scientific, model: Rocker Gyratory Lab Scale TSSL3, catalog number: 51900-27 )
  4. Thermocycler
  5. Microbiological incubator with 37 °C, atmospheric CO2 (Thermo Fisher Scientific, Heraeus)
  6. HeraCell 150 incubator with 37 °C, 5% CO2 (Thermo Fisher Scientific, model: HeraCell 150 incubator )
  7. MUPID-One electrophoresis system (LABGENE Scientific, model: MUPID-One electrophoresis system )
  8. Bench-top centrifuge (Eppendorf, model: 5417 C )
  9. Nalgene Oak Ridge centrifuge tube (Lab Depot, catalog number: 21009-386-PK )
    Note: These tubes are appropriate for ultracentrifugation and can’t be exchanged by standard tubes.
  10. Water bath (Thermo Fisher Scientific)
  11. Thermomixer comfort (Eppendorf)
  12. Vortex mixer Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 )
  13. FACSARIA III flow cytometry sorter (BD, model: FACSAria III )
  14. FACS Canto II flow cytometer (BD, BD Biosciences, model: FACSCANTO II )
  15. Gel documentation (Syngene)
  16. Heraeus Multifuge 3S (Thermo Fisher Scientific, model: HeraeusTM MultifugeTM 3S )
  17. Laminar flow hood for sterile tissue culture, biosafety level 2 approved (Thermo Fisher Scientific)
  18. Mastercycler nexus (Eppendorf)
  19. Mr. Frosty freezing container (Thermo Fisher Scientific)
  20. Neubauer chamber (BRAND)
  21. TransBlot Turbo transfer system (Bio-Rad Laboratories, model: Trans-Blot® TurboTM transfer system )
  22. Western blot chamber (Criterion Cell) and PowerPac basic power supply (Bio-Rad Laboratories)
  23. Spatula

Software

  1. A Plasmid Editor (http://biologylabs.utah.edu/jorgensen/wayned/ape/) (Optional)
  2. Oligonucleotide Properties Calculator (http://biotools.nubic.northwestern.edu) (Optional)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Behrmann, L., McComb, S., Aguadé-Gorgorió, J., Huang, Y., Hermann, M., Pelczar, P., Aguzzi, A., Bourquin, J. P. and Bornhauser, B. C. (2017). Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9. Bio-protocol 7(7): e2222. DOI: 10.21769/BioProtoc.2222.

ris ris Download Citation in RIS Format

分类

分子生物学 > DNA > 诱/突变

细胞生物学 > 细胞分离和培养 > 细胞分离

细胞生物学 > 细胞工程 > CRISPR-cas9

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件