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Jun 2020

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Conditional Human BRD4 Knock-In Transgenic Mouse Genotyping and Protein Isoform Detection
条件性人 BRD4 敲入转基因小鼠基因分型和蛋白质异构体检测   

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Abstract

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions.


Graphic abstract:



Keywords: BRD4 isoforms (BRD4 异构体), BET bromodomains (BET 溴结构域), Knock-in Transgenic mice (敲入转基因小鼠), Genotyping (基因分型), PyMT (PyMT), Breast cancer (乳腺癌), TNBC (TNBC), Western blotting (蛋白质印迹)

Background

Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra-terminal (BET) family proteins that also include BRD2, BRD3, and testes/germ cell-specific BRDT (Wu and Chiang, 2007). BET proteins are epigenetic regulators implicated in chromatin dynamics and cancer development (Li et al., 2018; Kim et al., 2019; Wu et al., 2020). BRD4 is typically required for expression of c-Myc (Zuber et al., 2011), FosL1 (Lockwood et al., 2012), and other oncogenes, primarily via enhancer/promoter regulation (Lovén et al., 2013; Wu et al., 2020). With the availability of small-compounds inhibiting BET proteins, particularly BRD4 (Filippakopoulos et al., 2010; Nicodeme et al., 2010; Cai et al., 2011; Chiang, 2014; Tang et al., 2021; Liu et al., 2022), epigenetic therapy targeting BET proteins is closer to reality. Nevertheless, accurate detection of BET proteins in various cell lineages and tissues can be challenging, due to their tight association with chromatin in a confined nuclear compartment (Wu et al., 2006), dependency on BET protein phosphorylation for chromatin association and factor interaction (Chiang, 2016; Wu et al., 2013, 2016), and existence of multiple protein isoforms (Chiang, 2014; Wu et al., 2020). In humans, BRD4 has two universally expressed protein isoforms (Wu and Chiang, 2007) – BRD4-L (long, aa 1-1,362) and BRD4-S (short, aa 1-722) – generated by alternative 3′ splicing. Without proper solubilization and prompt processing of the isolated nuclear samples, BET proteins can be easily degraded or remain bound to chromatin, making it difficult to detect the intact proteins or distinguish the intrinsic smaller protein isoforms from the degradation products of the long isoform.


Using a conditional BRD4 knock-in (KI) transgenic mouse model, we showed that human BRD4-L plays a tumor suppressive role, and BRD4-S has oncogenic activity in breast cancer initiation, progression, and metastasis (Wu et al., 2020). BRD4-L and BRD4-S share common N-terminal amino acid (aa) residues from 1 to 719, with BRD4-S possessing three additional residues (GPA: glycine-proline-alanine) after aa 719, and BRD4-L containing a much longer C-terminal extension (Figure 1). To distinguish BRD4-S from BRD4-L, we have generated isoform-specific (S and L) and common (Pan) BRD4 antibodies. All of these are raised in rabbits, with a 14-aa peptide (S) or purified protein fragments (N and L) as antigens (Figure 1; for detail, see Wu et al., 2006 and 2016).



Figure 1. Human BRD4 protein isoform features and antibodies.

Abbreviations: BD1, first bromodomain; BD2, second bromodomain; NPS, N-terminal cluster of CK2 phosphorylation sites; BID, basic residue-enriched interaction domain; ET, extra-terminal domain; CTM, C-terminal motif. N is a pan antibody recognizing aa 149–284 commonly found in both BRD4-L and BRD4-S isoforms. Numbers correspond to the positions of the amino acid residues.


The BRD4-KI mice illustrated in this protocol are transgenic rodents, containing either a human BRD4-L or BRD4-S transgene introduced at the Rosa26 (Reverse orientation splice acceptor) locus (Figure 2A; Wu et al., 2020). Expression of the BRD4-L or BRD4-S transgene is controlled by the CAGGS promotor and a loxP-STOP-loxP cassette, where inducible expression of human BRD4 isoforms is triggered by Cre recombinase in a ubiquitous or tissue-specific manner. In the presence of Cre, recombination at the 34-bp loxP elements excises the STOP sequence, and allows the CAGGS promotor to drive BRD4 transgene expression (Figure 2A). Mice harboring human BRD4 transgenes can be crossed with other mouse strains to generate experimental and control groups for specific biological investigation. In this protocol, we utilize a breast cancer model in which BRD4-KI mice are crossed with mice expressing Cre and/or oncogenic PyMT (polyomavirus middle T antigen) in mammary fat pad tissue (see Table 1 for mouse strain information). Genomic DNA from mouse tail is then analyzed to genotype the offspring. The genotyping protocol starts with mouse tail snipping, tail lysis, polymerase chain reaction (PCR), and DNA agarose gel electrophoresis, to ultimately select for experimentation the mice with or without a specific transgene, namely human BRD4, MMTV-Cre, and MMTV-PyMT (Figure 2B). The Western blotting protocol details the important steps used to monitor endogenous mouse (m) and exogenous human (h) BRD4 protein expression, with emphasis on conditions for BRD4 solubilization from chromatin, use of wet protein transfer, and BRD4 isoform-specific (α-BRD4-L and α-BRD4-S) and Pan (α-BRD4-N) antibodies.



Figure 2. Schematic of human BRD4-L/S knock-in constructs and outline of genotypic and phenotypic analyses.

(A) Schematic drawing of hBRD4-L/S knock-in mouse models showing Cre-loxP-dependent recombination that triggers CAGGS promoter-driven expression of the FLAG-Myc-tagged BRD4-L/S transgene located in the Rosa26 gene locus in a ubiquitous or tissue-specific manner. (B) Flowchart detailing the experimental scheme involved in transgenic mouse genotyping from tail snipping and lysis, PCR, agarose gel electrophoresis, and mouse selection for studying PyMT-induced tumor growth with or without Cre-mediated BRD4 transgene expression and downstream phenotypic, protein, and histologic analyses. Abbreviation: MMTV, mouse mammary tumor virus.


The advantage of our genotyping procedure is the unique PCR protocol used to detect the presence of hBRD4-L and hBRD4-S in mice, using human allele-specific primers for amplification of the common or unique coding regions of each BRD4 isoform (Figure 3A for gene maps, and Table 2 for primer information). A PCR primer pair specific to hBRD4-L exons 13 and 15 (see Figure 3A, hBRD4-L, arrows for primer positions) is used to amplify the hBRD4-L transgene, which produces a PCR product of 216 bp (Figure 3B, hBRD4-L gel). To amplify hBRD4-S, we use a forward PCR primer which anneals to common hBRD4 exon 11, and a reverse primer unique to hBRD4-S exon 12 (see Figure 3A, hBRD4-S), yielding a PCR product of 136 bp (Figure 3B, hBRD4-S gel). A quick independent validation of either hBRD4 transgene is performed by using a forward primer that anneals to the FLAG sequence and a reverse primer that recognizes hBRD4 exon 3 (Figure 3A, hBRD4-Pan), producing a PCR product of 520 bp (Figure 3B, hBRD4-Pan gel). The human BRD4 transgene is inserted into the Rosa26 locus located on mouse chromosome 6. To amplify this region, we select a forward and reverse primer pair flanking a provirus integration site of the Rosa26 locus (Figure 3A; Soriano, 1999). Detection of a 600-bp PCR product representing an uninserted locus would indicate no BRD4 transgene, or a heterozygous configuration if concurrent BRD4-L or -S transgene is detected.


To detect the presence of the Cre transgene necessary for hBRD4-L/S expression, a Cre-specific primer pair (He et al., 2003; Wu et al., 2020) is used, generating a PCR product of 374 bp (Figure 3B, MMTV-Cre gel). With this primer pair, the zygosity of Cre cannot be determined; however, for Cre-mediated loxP recombination, a homozygous Cre genotype (Cre/Cre) is not essential (Reference 17). Sometimes, the Cre protein can cause unexpected phenotypes in certain heterozygous MMTV-Cre mouse strains (e.g., line A), such as lactational defects ( Yuan et al., 2011), or chromosomal abnormalities in some Cre-overexpressed mice, where DNA damage is induced in postmitotic spermatids possibly due to loxP-like sequence-triggered recombination in the mammalian genome (Schmidt-Supprian and Rajewsky, 2007). Thus, all Cre mouse strains used in this protocol are derived from line D (Wagner et al., 1997), and bred to carry only one copy of the Cre gene for tissue-specific expression, to avoid overexpression-induced defects. Since neither hBRD4-L or hBRD4-S alone is sufficient to induce mouse mammary tumors (our unpublished data), oncogenic PyMT is used to induce tumor formation in the mammary fat pads of female mice (Guy et al., 1992). PyMT exon 2-specific forward and reverse primers (Figure 3A) were used, resulting in a PCR product of 569 bp (Figure 3B, MMTV-PyMT gel). Hemizygous female MMTV-PyMT mice were purchased from the Jackson Laboratory, as they produce sizable mammary fat pad tumors for breast cancer study, so all mice in this protocol are bred to carry only one copy of the MMTV-PyMT gene.



Figure 3. Gene maps showing primer-annealing sites on specific transgenes and the Rosa26 locus, as well as the sizes of expected PCR products.

(A) Genomic maps of hBRD4-L, hBRD4-S, MMTV-PyMT, and MMTV-Cre transgenes and the Rosa26 locus. Positions of each PCR primer pair are shown as arrows above each gene map. (B) Agarose gel images showing the specificity of each primer pair and the size of its resulting PCR product. The presence (+) or absence (-) of a transgene, monitored by PCR, is shown in paired gel images (same exposure) from nonadjacent lanes (marked by a vertical dash line) in the same gel. The DNA ladders (in base pairs) are marked on the left. The bands representing primer dimerization (*) are also indicated.


Using the selective PCR strategies outlined above together with proper breeding schemes, hBRD4-KI genotyping can be easily performed to study the effects of conditional hBRD4 isoform expression on cancer initiation and tumor growth. Analysis of various downstream phenotypes, including tumor size/weight measurements, morphological hematoxylin and eosin (H&E) staining, and detection of isoform expression by Western blotting and immunohistochemistry (IHC), can then be carried out (Figure 2B). Critical steps, including the use of a high-salt protein extraction buffer, wet transfer, and BRD4 isoform-specific/Pan antibodies for detection of BRD4 protein isoforms (BRD4-L ~200 kDa and BRD4-S ~120 kDa), are highlighted in the accompanying Western blot procedure. When compared to semi-dry protein transfer, our protocol provides consistently better quality and reliability of high-molecular-weight protein transfer, with a much reduced background.

Materials and Reagents

  1. Acetic Acid, Glacial, ACS grade (Fisher Scientific, catalog number: A38C-212)

  2. Agarose Powder (Research Products International, catalog number: A20090-500.0)

  3. Aprotinin (Fisher Scientific, catalog number: BP250310)

  4. BRD4 Antibodies (in-house; described in Wu et al., 2006 and 2016)

  5. α-Cre Antibody (Millipore, catalog number: 69050-3)

  6. Direct PCR Lysis Reaction (Tail) Solution (Viagen, catalog number: 102-T)

    NOTE: Do not use Direct PCR Lysis Reaction solution that has been open for more than one year. After this period, precipitates appear, and efficiency decreases significantly due to incomplete tail tissue lysis.

  7. DNA Ladder, 1 kb Plus (Thermo Scientific, GeneRuler, catalog number: SM1333)

  8. Dry Ice

  9. Ear Tags (National Band and Tag Company, catalog number: 1005-1P)

  10. ECL Detection Reagents:

    West Femto Chemiluminescent Substrate (Thermo Scientific, SuperSignalTM, catalog number: PI34095)

    ECL HRP Substrate (Advansta, WesternBright®, catalog number: K-12045)

    Western Blotting Detection Reagents (Millipore-Sigma, ECLTM, catalog number: GERPN2209)

  11. Ethidium Bromide, Molecular Biology grade (Sigma, catalog number: E7637-5G)

  12. Ethyl Alcohol, 200 Proof (Pharmco, catalog number: 111000200)

  13. Ethylenediaminetetraacetic Acid Sodium Salt (EDTA, Research Products International, catalog number: E57020-500.0)

  14. Filter Paper:

    Pure Cellulose Chromatography Paper (Fisher Scientific, FisherbrandTM, catalog number: 05-714-4)

    Thick Blot Filter Paper, Precut, 7.5 × 10 cm (Bio-Rad, catalog number: 1703932)

  15. Gel Loading Dye, Purple, 6× (New England BioLabs Inc., catalog number: B7024S)

  16. Glycine (Fisher Scientific, catalog number: BP381-5)

  17. Isopropyl Alcohol (2-Propanol, Fisher Scientific, catalog number: A416-4)

  18. Labeling Tape, Self-Sticking (Fisher Scientific, catalog number: 1590110R)

  19. Leupeptin (Fisher Scientific, catalog number: 50-114-6410)

  20. Liquid Nitrogen

  21. Methanol (Pharmco, catalog number: 339000000)

  22. Mice (see Table 1)


    Table 1. Mouse strains used in this protocol.


  23. Microcentrifuge Tubes, 1.5 mL, Autoclaved (USA Scientific, catalog number: 1415-2500)

  24. Nitrocellulose Membranes, Supported, 0.2 mm (Fisher Scientific, Cytiva AmershamTM ProtranTM, catalog number: 45-004-017)

  25. NP-40 (Thomas Scientific, catalog number: C953H73)

  26. PCR Primers (Sigma-Aldrich)
    NOTE: See Table 2 for information on each primer sequence, annealing site, and product size.

    Table 2. Primers used for mouse tail PCR reactions.
    The databases, nucleotide sequences, and expected PCR sizes are indicated.

  27. PCR Tubes, Dome Strip Caps, 0.2 mL, 8-Strip (Phenix Research Products, catalog number: MPX-445)

  28. Pepstatin A (Fisher Scientific, catalog number: 501146412)

  29. Phenylmethylsulfonyl Fluoride (PMSF, Sigma, catalog number: P7626)

  30. Phosphatase Inhibitor Cocktail 2 (Sigma, catalog number: P5726)

  31. Phosphate-Buffered Saline (PBS), pH 7.4Plastic Food Wrap (Kirkland Signature, catalog number: 208721)

  32. Pre-Stained Protein Standards: Dual Color Standards (Bio-Rad, Precision Plus ProteinTM, catalog number: 1610374)

  33. Duo Pre-Stained Protein Ladder (Li-Cor Biosciences Inc., Chameleon®, catalog number: 92860000)

  34. Multicolor Broad Range Protein Ladder (ThermoFisher, SpectraTM, catalog number: 26634)

  35. Protein Assay Dye Reagent Concentrate (Bio-Rad, catalog number: 500-0006)

  36. Protein Gels, 4–20%, 15 Well, 15 µL (Bio-Rad, Mini-PROTEAN® TGX Stain-FreeTM, catalog number: 4568096)

  37. Anti-Rabbit Antibody Conjugated with Horseradish Peroxidase (HRP) or Fluorescence:Goat Anti-Rabbit IgG Secondary Antibodies (Southern Biotechnology, catalog number: OB4050-05)

  38. Anti-Rabbit IgG, 800 4× PEG Conjugate (Cell Signaling Technology, DyLightTM, catalog number: 5151)

  39. Reactive Brown 10, Practical grade (Sigma-Aldrich, catalog number: R0385-25G)

  40. Skimmed Milk Powder (Nestlé S.A., Carnation®)Sodium Chloride (NaCl, Fisher Scientific, catalog number: BP358-10)

  41. Sodium Deoxycholate (Fisher Scientific, catalog number: BP349-100)

  42. Sodium Dodecyl Sulfate (SDS, Fisher Scientific, catalog number: BP166-500)

  43. Sodium Hydroxide Pellets, ACS grade (Fisher Scientific, catalog number: S318-3)

  44. Tin Foil (Reynolds Wrap, catalog number: 2866C)

  45. Tissue Homogenizing Tubes, 2 mL (Bertin Corp., CKMix, catalog number: P000918LYSK0A.0)

  46. Tris Base (Research Products International, catalog number: T60040-5000.0)

  47. α-β-Tubulin (H-235) Antibody (Santa Cruz Biotechnology, catalog number: sc-9104)

  48. Tween 20 (Research Products International, catalog number: P20370)

  49. Universal SYBR® Green Supermix, 2× (Bio-Rad, SsoAdvancedTM, catalog number: 1725274)

  50. Water (Milli-Q®)Water, Nuclease-Free (Ambion, catalog number: AM9937)

  51. X-Ray Film (Fisher Scientific, Cytiva AmershamTM HyperfilmTM ECL, catalog number: 45-001-508)

  52. 1× SDS-PAGE Buffer (see Recipes)

  53. 1× Wet Transfer Buffer (see Recipes)

  54. 20× TBS-T Buffer (see Recipes)

  55. EDTA, 0.5 M, pH 8.0 (see Recipes)

  56. Ethanol, 70% (see Recipes)

  57. Ethidium Bromide Solution, 5 mg/mL (see Recipes)

  58. mRIPA Buffer, 400 mM (see Recipes)

  59. PCR Primer Working Solutions, 5 µM (see Recipes)

  60. Proteinase K Solution, 20 mg/mL (see Recipes)

  61. Tris-Acetate-EDTA Buffer, 1×, pH 8.3 (TAE, see Recipes)

Equipment

  1. AC Imaging System (Ultra-Violet Products Ltd., BioSpectrumTM)

  2. Analytical Balance (Mettler Toledo, catalog number: PG5002-S)

  3. Centrifuge (Beckman Coulter, AllegraTM X-22R, catalog number: 392187)
    NOTE: The S2096 rotor attachment is compatible with 0.2 mL PCR tubes.

  4. Centrifuge (Eppendorf, 5424, catalog number: EP-022620401)
    NOTE: The FA-45-24-11 rotor attachment is compatible with the 1.5 mL microcentrifuge tubes.

  5. Ear-Tag Applicator, Stainless (Braintree Scientific, catalog number: EP1005S1)

  6. Electrode Power Cables, Black & Red (Bio-Rad, Sub-CellTM GT)

  7. Freezers, -20°C and -80°C Gel Combs, 15-Well, Fixed Height, 0.75 Thickness (Bio-Rad, Sub-CellTM GT, catalog number: 1704445)

  8. Gel Eletrophoresis Tank (Bio-Rad, DNA Sub-CellTM)

  9. Gel Imaging System for Fluorescence or Chemiluminescence: Odyssey Infrared Imaging System (Li-Cor Biosciences Inc., 9120)

  10. Tabletop Film Processor (Konica Minolta, model: SRX-101A)

  11. Gel Trey, UV-Transparent, 15 × 25 cm (Bio-Rad, Sub-CellTM GT, catalog number: 1704419)

  12. Homogenizer (Bertin Corp., PreCellys® Evolution, catalog number: P000062-PEVO0-A)

  13. Magnetic Stir Bar, Octagon (The Lab Depot, SpinBar®, catalog number: 58948-171-EA)

  14. Magnetic Stirrer with Hot Plate (Fisher Scientific, IsoTempTM, catalog number: 11-100-49SH)

  15. Microwave (Kenmore)

  16. Mouse Dissection Tools

  17. pH Meter (Fisher Scientific, FisherbrandTM Accumet Basic and BioBasicTM, catalog number: 13-636-AB15)

  18. Power Supply (Bio-Rad, PowerPac 200)

  19. Refrigerator, 4°CRoller (Bio-Rad, catalog number: 1651279)

  20. Sonic Dismembrator (Fisher Scientific, FisherBrandTM, catalog number: FB120)

  21. Tetra Vertical Electrophoresis Cell for Mini Precast Gels (Bio-Rad, Mini-PROTEAN®, catalog number: 1658005)

  22. Thermal Cycler (Bio-Rad, T100TM Thermal Cycler)

  23. Tweezers (Fisher Scientific, FisherbrandTM, catalog number: 12-000-123)

  24. Vortexer (Scientific Industries Inc., Vortex-Genie 2 – 6560, catalog number: SI-0236)

  25. Water Bath (Fisher Scientific, IsoTemp 220TM, catalog number: 15-462-20)

  26. Wet Protein-Transfer Apparatus (Bio-Rad, Mini Trans-Blot Module, catalog number: 1703935)

Software

  1. Adobe Photoshop 2020 (Version 21.2.4)

  2. ImageJ (Version 1.53)
    NOTE: Image J may be used instead of Adobe Photoshop to analyze the agarose gel and Western blot images.

  3. Li-Cor Image Studio (Version 5.2)

  4. Microsoft Office Professional Plus (Excel)

  5. Microsoft Office Professional Plus (PowerPoint)

  6. UVP VisionWorksLS Image Acquisition and Analysis Software (Version 7.0.1)

Procedure

Part I: Genotyping Procedure
NOTE: A downloadable Mouse Genotyping Excel spreadsheet containing all the tables needed for organization and calculations in this procedure is attached to this protocol.

  1. Mouse Tail Tissue Acquisition – Day 0 
    A basic level of mouse maneuvering and husbandry abilities (e.g., scruffing, handling and restraining, sexing, mouse ear tagging, among others) is needed before performing this procedure.
    1. Start genotyping the mice around 2–3 weeks after birth, according to the regulations of your institutional ethics committee on animal experimentation.


      Figure 4. MMTV-Cre mice have a light-yellow fur color due to a physically linked K14-Agouti cassette which codes for a 131-aa protein regulating pheomelanin (yellow pigment) production (Hustad, 1995).

    2. Decide which mice to genotype. NOTE: Typical Mendelian inheritance is observed with all genes mentioned in this procedure. Thus, a Punnett square is used to predict the genotypes of any potential offspring from each breeding pair, if the genotypes of its parents are already known. Mice that do not come from parents that are homozygous for a gene typically need to be genotyped, except for MMTV-Cre offspring, which exhibit a “yellow” coat when crossed with a mouse in a B6-WT background due to the K14-Agouti transgene that is jointly inherited with the MMTV-Cre gene (Yuan et al., 2011). Thus, these mice can be visually selected solely based on the color of their fur (Figure 4).


      Figure 5. Mouse tail tissue acquisition.
      (A) Microscissors, tweezers, and an ear-tag applicator. (B) Styptic powder. (C) Labeled PCR tubes. (D) Restrained and scruffed mouse with ear tag. (E) Length of a typical mouse tail. (F) Mouse tail snipping. (G) Length of a 2-mm mouse tail tissue sample. (H) Mouse tail covered with styptic powder. (I) Harvested mouse tail-tip sample.

    3. Tag the mice, if not already tagged (Figure 5A and 5D). 
    4. Label a set of PCR tubes with each corresponding mouse’s tag number (Figure 5C). 
    5. Restrain and scruff the mouse (Figure 5D). 
    6. Using sharp surgical scissors, snip approximately 2 mm of the tail (Figure 5A, 5E–5G).
      Note: No more than 5 mm of the tail should be removed from each animal during its entire lifetime (see the ruler in Figure 5E, and the tail length in Figure 5G). Anesthesia should be used while obtaining tail-tip tissue from mice older than 28 days.
    7. Apply styptic powder to the end of the mouse’s tail to reduce bleeding (Figure 5B and 5H). 
    8. Return the mouse to its cage.
    9. Use tweezers to place the tail clipping directly into its corresponding PCR tube (Figure 5I). 
    10. Disinfect the surgical scissors with isopropyl alcohol.
    11. Repeat the above steps for all mice that need to be genotyped. 
    12. Place the mouse tail samples at 4°C, if they are not going to be lysed immediately; if longer than a day, store them at -20°C for up to six months (avoid multiple freeze-thaw cycles). 

  2. Prepare Lysis Reaction Working Solution – Day 1 
    1. For a 2-mm mouse tail tissue, 70 µL of Direct PCR Lysis Reaction (Tail) solution (see step C1 below) containing 0.4 mg/mL Proteinase K (see Recipes) is needed for each 0.2 mL PCR tube.
    2. Calculate the total amount of Direct PCR Lysis Reaction solution (Lysis Rxn Sol.) needed to lyse all the tail samples.
      e.g., 70 µL × 8 samples = 560 µL of Direct PCR Lysis Reaction Solution
      NOTE: Do not use Direct PCR Lysis Reaction solution that has been open for more than 1 year. Proteinase K is stable in Direct PCR Lysis Reaction solution for approximately 24 h.
    3. For every 1 mL of Direct PCR Lysis Reaction solution, 20 µL of Proteinase K solution is needed. Calculate the total amount of Proteinase K solution needed, based on the total amount of Direct PCR Lysis Reaction solution.
      NOTE: If frozen Proteinase K solution is used, thaw the solution in a 37°C water bath and invert intermittently to suspend the precipitated SDS into solution. Wait until the SDS has been re-dissolved into solution – which will become clear – before adding it to the Direct PCR Lysis solution.
      e.g., (560 µL Lysis Rxn Sol.)((1 mL Lysis Rxn Sol.)/(1000 µL Lysis Rxn Sol.))((20 µL Proteinase K Sol.)/(1 mL Lysis Rxn Sol.))=11.2 µL of Proteinase K Sol. 
    4. Add the total amount of Proteinase K solution calculated above to the total amount of Direct PCR Lysis Reaction solution to prepare the Lysis Reaction Working solution.


  3. Lyse the Tail-Tip Tissues – Day 1
    1. Add approximately 70 µL of the Lysis Reaction Working solution to each snipped mouse tail.
    2. Incubate each sample in the thermal cycler at 55°C for 12 h (step 1), followed by 85°C for 45 min (step 2), and then hold at 4°C or 12°C (depending on the default temperature for the specific PCR machine, step 3).
      Note: Complete tail tissue lysis is important. If some tail tips are not fully in contact with the solution, reposition the samples by gently tapping each PCR tube. The lysed tail samples can also be kept at 4°C overnight before PCR analysis.

  4. Process Crude Tissue Lysate – Day 2 
    1. Take the samples out of the thermal cycler and precipitate any remaining debris by centrifugation at 123 × g for 1 min.
    2. Label a new set of 1.5 mL microcentrifuge tubes with each mouse’s tag number.
    3. Transfer the crude lysate to the labeled microcentrifuge tubes.
      Note: Make sure not to pipette any of the mouse tail tissue debris with the lysate.
    4. If not immediately used, store the lysates at 4°C for up to one week, or at -20°C for up to six months.
      Note: Some of the components in the Direct PCR Lysis Reaction solution may interfere with PCR, especially during hBRD4-S gene amplification. To increase the PCR product yield, particularly for hBRD4-S, the crude lysate can be diluted 10-fold with nuclease-free water and then used for PCR. If PCR fails, the stored crude lysate solution can be used to re-start the procedure from this step.

  5. Primer Selection – Day 2 
    1. Decide the total number of PCR samples needed to genotype each mouse. 
    2. Refer to Section A2 regarding which mice need to be genotyped. These criteria will also dictate which genes need to be confirmed by PCR.
    3. In general, mice that do not come from parents that are homozygous for a gene will need to be genotyped for the presence of that gene; the exception being mice that have “yellow” coats indicating the presence of the MMTV-Cre gene (see Figure 4; Yuan et al., 2011).
    4. To properly determine the zygosity of the BRD4-L and BRD4-S transgenes (if both the parents are not homozygous for either hBRD4 isoform, and the offspring have the potential of being heterozygous for either BRD4 transgene allele), PCRs with BRD4-L or -S primer pairs are used to confirm knock-in, and then further validated by another PCR with the Rosa26 primer pair. Interpretation of the Rosa26 PCR results is as follows:
      1. For mice with homozygous hBRD4-L/L, hBRD4-S/S, or heterozygous hBRD4-L/S, no Rosa26 DNA band will appear in the agarose gel when the Rosa26 primer pair is used (Figure 3B, Rosa26 gel).
      2. For mice without BRD4-KI, or heterozygous Rosa26 mice with one copy of the BRD4 transgene, a Rosa26 PCR band will appear in the agarose gel when the Rosa26 primer pair is used (Figure 3B, Rosa26 gel).
    5. An optional BRD4-Pan primer pair can be used to confirm/exclude the presence of either BRD4 transgene, along with the Rosa26 primer pair (Figure 3B, hBRD4-Pan gel).
    6. PCRs with the MMTV-PyMT primer pair (Reference 18) are always included to confirm the presence of the MMTV-PyMT gene, because all our PyMT breeders carry only one copy of the MMTV-PyMT gene (Figure 3B, MMTV-PyMT gel).
    7. Positive and negative control samples with known genotypes need to be included for each PCR primer pair.

  6. Master Mix Solution Preparation – Day 2
    1. The Master Mix solution for all primers is a mixture of 7.5 µL of 2× SYBR® Green mix, 1.5 µL of the respective PCR Primer Working solution (at a concentration of 5 µM; see Recipes), and 5 µL of nuclease-free water, for a total volume of 14 µL of Master Mix solution in each PCR tube (Table 3).
      Note: The reagents used to make the Master Mix are relatively unstable and should always be kept on ice. To avoid contamination, the Master Mix should never be prepared directly from the original reagent containers, but rather from an already aliquoted secondary container. It typically takes about 30 min to thaw all the reagents on ice. After thawing, remember to always vortex (or invert) before aliquoting.

      Table 3. Sample Mouse Genotyping Matrix illustrating one way to organize the total number of PCR tubes needed to genotype several different types of transgenic mice.

      In this example, 8 transgenic mice are being genotyped. The green font indicates the PCR tube is optional.
      Note: The BRD4-Pan primer pair can be used to confirm the presence of the BRD4-L and/or BRD4-S isoforms. In general, the MMTV-Cre PCR tube is optional, as mice carrying the MMTV-Cre gene exhibit a “yellow” coat when crossed with a mouse in a B6-WT background (Yuan et al., 2011) and thus can be visually selected without the need for genotyping (see Figure 4).

    2. Calculate the amounts of each reagent needed to prepare each respective Master Mix, as shown in Table 3. Remember to consider approximately 10–30% more solution volume for a margin of error.
    3. To aid in organization and sample calculation, a mouse genotyping Excel spreadsheet is set up in Microsoft Excel as shown in Table 4, with the PCR Master Mix calculations illustrated in Table 5.

      Table 4. Mouse Genotyping Matrix spreadsheet.


      Table 5. PCR Master Mix spreadsheet for sample calculation.


  1. Total up the amounts needed for each reagent, and verify that all reagents are present in sufficient quantities before advancing.

  2. Prepare each Master Mix solution.

  3. Label a new set of PCR tubes with the corresponding tube numbers from the Mouse Genotyping Matrix (Table 3).

  4. For each primer set, add 1 µL of crude lysate to the bottom of each PCR tube, followed by the addition of 14 µL of Master Mix solution. This order of addition helps distinguish added from unadded samples.

  5. Repeat the same steps for all other primer sets.

  6. Store any remaining crude lysate at -20°C for up to six months.

    Note: If PCR fails, the stored crude lysate solution can be used to re-start the reaction. Making aliquots helps avoid sample degradation caused by multiple freeze-thaw cycles.

  7. Centrifuge the samples at 123 × g for 1 min in the Allegra X-22R centrifuge at room temperature (RT).


  1. Polymerase Chain Reaction (PCR) – Day 2

    1. For BRD4 transgene detection, set the PCR protocol to 94°C for 3 min (step 1), 94°C for 10 s (step 2), 60°C for 30 s (step 3), and 72°C for 30 s (step 4). Repeat steps 2–4 for 35 cycles. Finish the protocol sequence at 72°C for 3 min, and then hold at 4–12°C.

      Note: Step 1 – 94°C for 3 min – is to increase DNA denaturation and heat-activate the Sso7d fusion DNA polymerase included in the Universal SYBR® Green Supermix.

    2. Load all PCR tubes for BRD4 transgene amplification into the thermal cycler.

    3. Press Run, set the PCR volume to 15 µL when prompted by the instrument, and then press OK.

    4. Perform PCR for the other samples using another thermal cycler with the settings as described in step G1, but repeat steps 2–4 for 40 cycles.

    5. This PCR procedure will take approximately 1.5 h to finish.

    6. If not for immediate use, place the finished PCR samples at 4°C for up to one week, or at -20°C for up to six months.

      Note: Sections H through N, included below for beginners, are not specific to this procedure and may be replaced by your own lab protocols.


  2. Prepare Agarose Gel Solution – Day 3

    1. Weigh out approximately 3.0 g of agarose, add it to 200 mL of 1× TAE buffer (pH 8.3, see Recipes), and stir to make a 1.5% agarose solution.

      Note: This recipe is intended for a 15 × 25 cm gel-casting tray.

    2. Heat the solution in a microwave for approximately 3 min, or until the agarose is fully dissolved.

    3. Add 15–20 µL of a 5 mg/mL solution of ethidium bromide (see Recipes) to the agarose solution.

    4. Let the solution stir at RT until lukewarm.


  3. Gel Casting – Day 3

    1. Use tape to cover the ends of the gel-casting tray, making sure to seal the outer edges.

    2. Evenly place four 15-well combs (for a total of 60 wells) into the gel-casting tray.

    3. Pour all the 1.5% gel solution into the gel-casting tray – enough so the solution reaches a height of 0.6–0.8 cm from the bottom.

    4. Remove any bubbles and evenly distribute the liquid.

    5. The gel takes approximately 20–25 min to set.


  4. Gel Loading – Day 3

    1. Remove the tape from each side of the gel-casting tray and place the tray in an electrophoresis tank.

    2. Remove the gel-casting combs and submerge the gel in 1× TAE buffer (pH 8.3). If not for immediate use, place the gel slab in 1× TAE buffer (see Recipes), and store at 4°C.

    3. Take the PCR samples from the refrigerator, and add 3 µL of 6× gel-loading dye to each sample.

    4. Load samples in groups, each with the 1 kb Plus DNA ladder in the beginning lane, and positive and negative controls in the last two lanes of each PCR primer group. Skip a well after each primer set and start loading the following set of samples. Normally, 8 µL of DNA ladder and 10 µL of each PCR sample is enough to produce prominent bands in the gel; however, this amount can vary depending on the amount of the PCR product produced.

    5. After loading the samples, connect the black/cathode (-) and red/anode (+) cables to the electrophoresis tank, and turn on the power supply. The current in the gel runs from cathode to anode (blackred) – the same direction the DNA samples migrate through the gel. Be careful not to switch the electrode power cables.

    6. Set the voltage (V) to 150 volts – the amperage (A) will then change to 2.00 A – and let the gel run for 30–45 min, or until the two dyes in the sample loading buffer are well separated.


  5. Gel Imaging – Day 3

    1. Take the gel out of the gel-casting tray and submerge it in distilled water.

    2. Turn on the BioSpectrum AC Image System, if not already on.

    3. Open the unit and clean the glass with 70% ethanol (see Recipes).

    4. Place the gel on the surface of the gel scanner and close the door.

    5. Launch the VisionWorks program and log in.

    6. Under the Lighting tab, turn the transillumination device onto UV light.

    7. Press Acquisition and Preview.

    8. Under the Lighting Tab, adjust the Zoom and Focus to sharpen the image.

    9. Under the Camera tab, adjust the exposure time (minutes and seconds) to change the intensity of the image.

    10. Press Capture to get the image.

    11. Under the Edit tab, rotate the images.

    12. Press Align to further adjust the angle, then crop the image.

    13. Click File and Save As.

    14. Save the image with the following format: DD MM YY, Initials, Mouse Genotyping Gel #.


  6. Gel Disposal – Day 3

    Due to the toxicity of ethidium bromide, any gel containing this chemical should be disposed of in an approved waste container.


  7. Gel Processing – Day 3

    1. Using Adobe Photoshop

      Note: ImageJ may be used instead of Adobe Photoshop to process the gel images.

      1. Open the scanned image in Adobe Photoshop. Under the Image panel, click on Image Rotation to properly orient the image. For 180 degrees, click 180°. For a particular angle, click Arbitrary.

      2. After proper orientation, click on the Image tab and then Mode; and click Greyscale.

      3. A pop-up box with Disregard Changes will come up, so click Discard.

      4. In the pane to the right, click Adjustments and select Invert.

      5. Toggle the contrast and brightness of the image under the Adjustments panel by clicking Brightness/Contrast.

      6. Save the image as a JPEG or TIFF file.

    2. Microsoft PowerPoint

      1. Place the black-and-white gel images from Adobe Photoshop into PowerPoint and label the gel lanes with the proper PCR tube numbers from the Mouse Genotyping Matrix (Table 3).

      2. Label the names of each primer set, positive control (P), negative control (N), and the molecular weight markers (M) where they correspond.


  8. Gel Interpretation – Day 4

    1. Refer to Figure 3B as a guide for proper band identification.

    2. Read the results for each PCR tube as laid out in the Mouse Genotyping Matrix (Table 3).

    3. Record the genotype of each mouse.


Tumor Dissection & Tissue Processing for Western Blotting

  1. Tumor Dissection

    1. Euthanize the tumor-bearing mouse (Figure 6A and 6B). Collect extra tail tissue for backup genotyping, and store at -20°C.

    2. Make a 5-mm horizontal incision at the top of the mouse’s abdomen, using microscissors to pierce the mouse’s skin (Figure 6C).

    3. Pull the skin below the incision caudally to expose the abdominal-pelvic cavity (Figure 6D).

    4. The tumors will be located in the fat pad tissue surrounding the peritoneum (Figure 6E).

    5. Remove the tumors from mammary fat pads #4 and #5, being careful not to take any of the surrounding tissue (Figure 6F).

      Note: Avoid rupturing any veins and arteries, because the bleeding will make the procedure difficult (see Figure 6E).

    6. Now pull the remaining skin above the primary incision cranially, until exposing the entire thoracic cavity (Figure 6G).

    7. The tumors will be located in the fat pad tissue attached to the posterior side of the skin flap (Figures 6H, I, and J).

    8. Remove the tumors from mammary fat pads #3, #2, and #1, respectively, being careful not to take any of the surrounding tissue (Figure 6K).

      Note: Avoid rupturing any veins and arteries because the bleeding will make the procedure difficult (see Figure 6G).

    9. Use an analytical balance to record the individual tumor weight and the aggregate tumor weight for all the mammary fat pad tumors (see Figure 6K).

    10. Place each of the tumors in labeled microcentrifuge tubes (with mouse tag number, mammary fat pad location, date, and sample type). If the tumor is too large to fit into the microcentrifuge tube, cut it into pieces and place it in the tube.

    11. Submerge the sample microcentrifuge tubes in liquid nitrogen to snap freeze the samples, and store them at -80°C.



    12. Figure 6. Mouse mammary PyMT tumor acquisition.

      (A) CO2 euthanasia system. (B) Euthanized mouse with mammary fat pads #1–5 labeled. (C) Mouse with incision located at its midline. (D) Mouse with a skin flap below the midline incision pulled caudally to reveal the peritoneum. (E) Abdominopelvic tumors. (F) Harvested PyMT tumors from mammary fat pads #4 and #5. (G) Mouse with a skin flap above the midline incision pulled cranially to reveal the thoracic cavity. (H) Mouse with the superior anatomical right side exposed to reveal mammary fat pad tumors #1–3. (I) Mouse with the superior anatomical left side exposed to reveal mammary fat pad tumors #1 and #2. (J) Mouse with the superior anatomical far left side exposed to reveal mammary fat pad tumors #2 and #3. (K) Total mammary fat pad tumors harvested. Abbreviation: MFP, mammary fat pad; L, left; R, right.


  2. Tumor Processing

    1. Take the frozen samples out of -80°C and place them in dry ice.

    2. Transfer the frozen tissue to a CKMix tissue homogenization tube (2 mL), and add mRIPA (400 mM) buffer (see Recipes – important to properly dissociate BRD4 from chromatin).

      Note: Add approximately 0.2–0.3 mL of mRIPA (400 mM) solution to each 40–60 mg tumor sample, enough to immerse the whole tissue.

    3. Homogenize the tumor tissues with the PreCellys Evolution Tissue Homogenizer.

      1. Turn on the PreCellys Evolution Tissue Homogenizer.

      2. Fill the top of the machine with dry ice and close the lid.

      3. Go to the Home Screen and select the Soft Protocol (with the following specifications: Tube Volume – 2 mL; Rotation Speed – 5,800 rpm; Cycle – 2 × 15 s; Pause – 30 s; Cryolysis – On; Temperature – 4°C; and Mode – Auto), Start, and press OK.

        Note: If a pressure error occurs, take all the samples out of the instrument and run the protocol again without samples; this should de-pressurize the machine. Alternatively, remove the dry ice chamber atop the machine and shake it, allowing the machine to de-pressurize.

      4. Make sure the samples are fully homogenized (i.e., no large tumor chunks are visible in the liquid). If not, run the Soft Protocol again.

      5. Once finished, leave the lid on halfway to let the dry ice evaporate in the cooling chamber, and turn off the machine.

    4. Place the samples on ice.

    5. Transfer the samples to a set of labeled, 1.5-mL microcentrifuge tubes. Take as much liquid as possible from each homogenized sample.

      Note: Be careful not to transfer any of the beads or residual mammary fat to the new sample tubes, as this can interfere with assay performance.

    6. Centrifuge the samples at 20,000 × g and 4°C for 30 min.

    7. After centrifugation, transfer at least 50 µL of supernatant to a second set of labeled tubes. Make additional 50–100 µL aliquots as needed.

    8. Keep one sample aliquot for each tumor sample to determine the protein concentration, snap freeze the remaining sample aliquots in liquid nitrogen, and store them at -80°C.

    9. Perform a Bradford assay to measure the approximate protein concentration of each sample aliquot. For this, prepare a 1x solution from the Bio-Rad Protein Assay Dye Reagent concentrate (5×) according to the manufacturer’s instructions.

      Note: For cell line samples, ~1 × 106 cells are washed with 1× cold PBS and lysed in a 2–5 fold volume of mRIPA (400 mM; see Recipes) buffer, and then mildly sonicated at 4°C (e.g., 3 pluses of 20% amplitude with a 1/8” microtip) to shear the genomic DNA and thus reduce viscosity for easy gel loading. The protein concentration of the cell lysate is determined by using the 1× Bio-Rad Protein Assay Dye Reagent.

    10. For the Bradford assay calibration curve, prepare BSA protein standards with the 1× Bradford dye.


Western Blotting Procedure

  1. Prepare 25–50 µg protein samples, from either cell line (see the preceding Tumor Processing section Note in Step B9), or PyMT tumor that has been homogenized using mRIPA (400 mM) buffer. For normal tissues, ~50 µg of protein sample are needed for endogenous BRD4-S detection.

  2. Cast 8% SDS-PAGE gels, or use 4–20% pre-made gradient gels.

  3. Load 5–10 µL of 5 mg/mL protein samples and pre-stained standards (usually 5 µL, depending on company sources).

  4. Perform gel electrophoresis. When using in-house 8% gels, start at 100V and then increase to 200V, once the pre-stained standards enter the resolving gel. Follow the manufacturer’s instructions for running pre-made gels.

  5. Cool the 1× Wet Transfer buffer (see Recipes) down to 4°C during gel running (important).

  6. Perform wet transfer using the Bio-Rad Mini Trans-Blot Module:

    When assembling the protein-transfer apparatus, be careful not to create bubbles in the 1× Wet Transfer buffer, as these will interfere with protein transfer and could possibly prevent proteins from transferring completely.

    1. Place the transfer cassette case on its clear side.

    2. Submerge one thin wet sponge in cold 1× Wet Transfer buffer and place it in the middle of the cassette.

    3. Next, submerge one thick piece of filter paper in cold 1× Wet Transfer buffer and place it on top of the sponge, making sure to center them as best as possible. Cut as needed.

    4. Use a roller to remove any air bubbles from the filter paper.

    5. Place one sheet of nitrocellulose membrane immersed in cold 1× Wet Transfer buffer on top of the filter paper and remove any air bubbles.

    6. Place the SDS-PAGE gel on top of the nitrocellulose membrane and remove any air bubbles.
      Note: Try to orient the SDS-PAGE gel as best as possible on the nitrocellulose membrane, being careful not to accidentally tear the gel. The best way to place the gel onto the nitrocellulose membrane is to let it swim atop the membrane in the 1× Wet Transfer buffer – in the liquid, the gel moves more freely. From there, jointly place both the membrane and the gel on the filter paper.

    7. Layer one more piece of thick filter paper immersed in cold 1× Wet Transfer buffer on top of the gel and remove any air bubbles.

    8. Finally, place one thin sponge immersed in cold 1× Wet Transfer buffer on top of the filter paper and close the cassette sandwich.

    9. Place the cassette in the apparatus so the black side of the cassette faces the black side of the protein transfer apparatus.

    10. Repeat the previous steps for any subsequent gels and then place an ice pack next to the black side of the protein transfer apparatus.

    11. Top off the protein transfer apparatus with any remaining cold 1× Wet Transfer buffer and place the apparatus at 4°C.

    12. Place the lid on top of the transfer apparatus and connect it to the power supply.

    13. Turn on the power supply and set the voltage to 50 V for 720 min (important).

    14. Press the Run button to start the wet protein transfer.

      Note: If done correctly, bubbles should be seen rising from the bottom of the buffer solution and the amperage should be around 0.25–0.30 Amp for two Western blots and 0.5–0.6 Amp for four Western blots.

    15. The apparatus will automatically turn itself off when finished.

  7. Remove the transfer device from 4°C and place each of the membranes in a solution of 5% skimmed milk diluted with 1x TBS-T buffer (see Recipes) at RT for 10 min.

    Note: Prior to milk blocking, an intermediate step can be included to check for the presence of bubbles that potentially block the transfer of protein from the gel to the membrane by brief staining (~2 min) of the blot in a 0.1% Reactive Brown 10 (or 0.01% Ponceau S) solution, followed by destaining with water. This additional step helps determine whether the experiment should be continued if most of the protein is not successfully transferred.

  8. Calculate the amount of each antibody and the diluent needed for dilution. Primary BRD4 antibodies: 1:2,000 for anti-BRD4-N (used to detect both BRD4-L and BRD4-S) and anti-BRD4-L antibodies, and 1:1,000 for the anti-BRD4-S antibody (e.g., add 3 µL of anti-BRD4-N antibody to 6 mL of 5% skimmed milk).

  9. Incubate the membrane in primary BRD4 antibody with gentle rocking at 4°C overnight (or 2 h at RT for detection of the more abundant BRD4-L protein).

  10. Following overnight incubation, take the blots out of 4°C, decant the 5% skimmed milk solution, and delicately remove any residual milk solution with four deionized water rinses.

  11. Decant the water from each blot, and replace it with 8–16 mL of 1× TBS-T buffer (see Recipes; the exact volume depends on the size of the chamber/tray used), allowing the membrane to float freely in the liquid.

  12. Let the blots rock at RT for 5 min.

  13. Wash all the blots with 1× TBS-T buffer, for a total of three times.

  14. Place the blots back in the 5% skimmed milk in 1× TBS-T solution, and add the secondary antibody at a 1:10,000–4,000 dilution (e.g., in 6 mL of 5% skimmed milk, add 0.6 µL of secondary antibody for a 1:10,000 dilution, and 1.5 µL of secondary antibody for a 1:4,000 dilution).

  15. Incubate the secondary antibody with gentle rotation on an orbital shaker at RT for 1 h.

  16. Decant the milk, and wash four times with deionized water, followed by four 1× TBS-T rinses, with a 5-min incubation in between as described in steps 10–13.

  17. After the final TBS-T wash, replace the solution with 1× PBS (pH 7.4), as this solution is more compatible with fluorescent signals (e.g., the Li-Cor Odyssey imaging system). Alternatively, add the ECL detection reagent, if using chemiluminescence detection by an X-ray film that is developed with the Konica tabletop film processor.

    Note: For low-abundant BRD4-S detection, use a high-sensitive ECL detection reagent, e.g., Thermo ScientificTM SuperSignalTM West Femto Chemiluminescent Substrate (Cat. #: PI34095).

  18. Representative images of BRD4 protein isoform detection can be found in Figure 7.



Figure 7. Western blots showing conditionally-induced human BRD4-S overexpression in three PyMT tumors from three representative Cre-expressing mice.

PyMT tumors from three mice with no Cre expression are included as controls. Protein size markers (in kDa) are indicated on the left of each gel image.

Recipes

  1. 5 µM PCR Primer Working Solutions

    To prepare a PCR Primer Working solution for each of the Master Mix solutions, as illustrated in Table 3, add 10 µL of the forward PCR primer (100 µM) to 10 µL of the reverse PCR primer (100 µM) in 180 µL of nuclease-free water, for each gene-specific primer pair.

  2. 0.5 M EDTA (pH 8.0)

    For a 1-L solution, add 186.1 g of EDTA to 800 mL of Milli-Q water. Stir vigorously on a magnetic stirrer and adjust the pH to 8.0 with NaOH pellets. Adjust the resulting solution to 1 L with Milli-Q water.

    Note: The solution may need to be heated slightly to completely dissolve all the EDTA powder.

  3. 1× Tris-Acetate-EDTA (TAE, pH 8.3) Buffer

    1. For a 20-L solution, add 96.8 g of Tris base, 22.84 mL of glacial acetic acid, and 40 mL of 0.5 M EDTA (pH 8.0) to enough deionized water for a final volume of 20 L.

    2. For a 1-L solution, add 4.84 g of Tris base, 1.14 mL of glacial acetic acid, and 2.0 mL of 0.5 M EDTA (pH 8.0) to enough deionized water for a final volume of 1 L.

  4. 5 mg/mL Ethidium Bromide

    Add 1 g of ethidium bromide to 200 mL of autoclaved/deionized water. Place the solution in a tin foil-wrapped glass container to avoid photodegradation, and store at 4°C.

    Note: Ethidium bromide is a mutagen. Any gels containing this chemical should be disposed of in an approved waste container.

  5. 70% Ethanol

    For a 1-L solution, add 700 mL of ethyl alcohol to 300 mL of Milli-Q water.

  6. Proteinase K Solution (20 mg/mL)

    To prepare 50 mL of Proteinase K buffer, add 20 mL of glycerol, 0.5 mL of 1 M Tris (pH 7.5), and 50 µL of 1 M calcium chloride to enough deionized water for a final volume of 50 mL. Dissolve 100 mg of Proteinase K powder (Fisher Scientific, Cat.#: BP1700-100) in 5 mL of Proteinase K buffer, and store the resulting solution at -20°C in 1-mL aliquots.

  7. mRIPA (400 mM) Buffer

    For a 50-mL solution of mRIPA (400 mM), mix together 2.5 mL of 1 M Tris-HCl (pH 7.5, final 50 mM), 4 mL of 5 M NaCl (final 0.4 M), 5 mL of 10% NP-40 (final 1%), 1.25 mL of 10% sodium deoxycholate (final 0.25%), 0.1 mL of 0.5 M EDTA (final 1 mM), and protease inhibitor (consisting of 0.5 µM of PMSF, 50 µg/mL of Leupeptin, 50 µg/mL of Aprotinin, 1 µg/mL of Pepstatin A, and a 500× dilution of Sigma phosphatase inhibitor cocktail 2).

  8. 1× SDS-PAGE Buffer

    1. For a 20-L solution, add 60 g of Tris base, 288 g of glycine, and 20 g of sodium dodecyl sulfate (SDS) to enough deionized water for a final volume of 20 L.

    2. For a 1-L solution, add 3 g of Tris base, 14.4 g of glycine, and 1 g of SDS to enough deionized water for a final volume of 1 L.

  9. 1× Wet Transfer Buffer

    For a 1-L solution, add 25 mL of 1M Tris-HCl (pH 8.0), 14.41 g of glycine, 1 g of SDS, and 200 mL of methanol to enough deionized water for a final volume of 1 L.

  10. 20× TBS-T Buffer

    For a 1-L solution, add 200 mL of 1 M Tris-HCl (pH 8.0), 175.32 g of NaCl, and 10 mL of 100% Tween 20 to enough deionized water for a final volume of 1 L.

Acknowledgments

We thank Dr. Chien-Fei Lee for technical help and guidance during protocol development and Claire Chiang and William Chiang for comments on the manuscript. The protocol detailed here is extended primarily from the procedures described in Wu et al. (2020). This work was supported in part by NIH grant 1RO1CA251698-01 and CPRIT grant RP190077.

Competing interests

The authors have no financial conflicts of interest.

Ethics

All animals are housed in a pathogen-free barrier facility with access to food and water ad libitum. All experiments are performed in accordance with Protocol # 2017-102140 approved by the UTSW Institutional Animal Care and Use Committee (IACUC).

References

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  26. Zuber, J., Shi, J., Wang, E., Rappaport, A. R., Herrmann, H., Sison, E. A., Magoon, D., Qi, J., Blatt, K., Wunderlich, M., et al. (2011). RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478(7370): 524-528.

简介

含溴结构域蛋白 4 (BRD4) 是一种乙酰赖氨酸读取蛋白和转录调节因子,与染色质动力学和癌症发展有关。已在人类中检测到几种 BRD4 同工型,其中长同工型 (BRD4-L, aa 1-1,362) 起肿瘤抑制作用,而主要的短同工型 (BRD4-S, aa 1-722) 在乳腺癌发展中具有致癌活性. BRD4 蛋白异构体的相反功能的体内证明需要开发小鼠模型,特别是有条件地表达人 BRD4-L 或 BRD4-S 的转基因小鼠,它们可以在不同的小鼠组织中以时空特异性方式选择性地诱导。在这里,我们详细介绍了用于对转基因小鼠品系进行基因分型的程序,这些程序旨在确定条件性人 BRD4 同种型表达对多瘤病毒中间 T 抗原 (PyMT) 诱导的小鼠乳腺肿瘤生长的影响,以及 BRD4 蛋白同种型的蛋白质印迹检测的关键步骤在那些肿瘤和培养的细胞中。以该协议为指导,BRD4 异构体功能的解释变得更加可行,并可扩展到各种生物环境。对 BRD4 亚型在体内和体外的分布进行充分跟踪是了解它们的生物学作用的关键,以及避免由于不正确使用无法检测其空间和时间分布的实验程序而对其功能的误解。


图形摘要:




背景

含溴结构域蛋白 4 ( BRD4) 是溴结构域和末端外 (BET) 家族蛋白的成员,该家族蛋白还包括 BRD2、BRD3 和睾丸/生殖细胞特异性 BRDT (Wu and Chiang, 2007)。 BET 蛋白是与染色质动力学和癌症发展有关的表观遗传调节因子(Li等人,2018;Kim等人,2019;Wu等人,2020)。 BRD4 通常是表达c - Myc (Zuber et al., 2011)、 FosL1 (Lockwood et al. , 2012) 和其他癌基因所必需的,主要通过增强子/启动子调节 ( Lovén) 等。 , 2013;吴等人。 , 2020)。随着抑制 BET 蛋白的小化合物的出现,特别是 BRD4 ( Filippakopoulos 等。 , 2010;尼科德美 等。 , 2010;蔡等人。 , 2011;蒋,2014;唐等人。 , 2021; Liu et al ., 2022),针对 BET 蛋白的表观遗传疗法更接近现实。然而,准确检测各种细胞谱系和组织中的 BET 蛋白可能具有挑战性,因为它们与受限核区室中的染色质紧密结合(Wu等,2006),染色质结合和因子相互作用依赖于 BET 蛋白磷酸化。 Chiang, 2016; Wu et al. , 2013, 2016),以及多种蛋白质亚型的存在 (Chiang, 2014; Wu et al. , 2020)。在人类中,BRD4 具有两种普遍表达的蛋白质同工型(Wu 和 Chiang,2007)——BRD4-L(长,aa 1-1,362)和 BRD4-S(短,aa 1-722)——由选择性 3 '剪接产生。如果不对分离的核样品进行适当的溶解和及时处理,BET 蛋白很容易降解或保持与染色质的结合,从而难以检测完整的蛋白质或区分内在较小的蛋白质同种型与长同种型的降解产物。
使用条件性 BRD4敲入 (KI) 转基因小鼠模型,我们发现人 BRD4-L 具有抑癌作用,而 BRD4-S 在乳腺癌的发生、进展和转移中具有致癌活性( Wu et al. , 2020 )。 BRD4-L 和 BRD4-S 在 1 到 719 之间共享共同的 N 端氨基酸 (aa) 残基,BRD4-S 在 aa 719 之后具有三个额外的残基(GPA:甘氨酸-脯氨酸-丙氨酸),而 BRD4-L 包含一个更长的 C 端扩展(图 1)。为了区分 BRD4-S 和 BR D4-L,我们生成了同种型特异性(S 和 L)和常见(Pan)BRD4 抗体。所有这些都是在兔子身上产生的,以 14-aa 肽 (S) 或纯化的蛋白质片段(N 和 L)作为抗原(图 1;详情参见 Wu等人,2006 和 2016)。



图 1. 人类 BRD4 蛋白亚型特征和抗体。
缩写:BD1,第一个溴域; BD2,第二个溴结构域; NPS,CK2磷酸化位点的N端簇; BID,基本残基富集的相互作用域; ET,终端外域; CTM,C 末端基序。 N 是一种泛抗体,可识别常见于 BRD4-L 和 BRD4-S 同种型中的aa 149 – 284。数字对应于氨基酸残基的位置。

本协议中说明的BRD4 -KI小鼠是转基因啮齿动物,含有在 Rosa26(R 逆向剪接受体)基因座处引入的人类BRD4 - L或BRD4 - S转基因(图2A ; Wu等人,2020 )。 BRD4 -L或BRD4-S转基因的表达由 CAGGS 启动子和loxP -STOP- loxP盒控制,其中人 BRD4 同种型的可诱导表达由 Cre 重组酶以普遍或组织特异性方式触发。在存在 Cre 的情况下,34 bp loxP元件的重组切除了STOP 序列,并允许 CAGGS 启动子驱动BRD4转基因表达(图 2A)。携带人类BRD4转基因的小鼠可以与其他小鼠品系杂交,以产生用于特定生物学研究的实验组和对照组。在该协议中,我们利用乳腺癌模型,其中BRD4-KI小鼠与在乳腺脂肪垫组织中表达 Cre 和/或致癌PyMT (多瘤病毒中间 T 抗原)的小鼠交叉(参见表 1 中的小鼠 stra信息)。然后分析来自小鼠尾巴的基因组 DNA 以对后代进行基因分型。基因分型方案从小鼠剪尾、尾部裂解、聚合酶链式反应 (PCR) 和 DNA 琼脂糖凝胶电泳开始,最终选择具有或不具有特定转基因的小鼠进行实验,即人类BRD4 、 MMTV-Cre和MMTV- PyMT (图 2B)。西方印迹协议详细介绍了用于监测内源性小鼠 (m) 和外源性人类 (h) BRD4 蛋白表达的重要步骤,重点是 BRD4 从染色质中溶解的条件、使用湿蛋白转移和 BRD4 亚型特异性(α- BRD4-L 和 α-BRD4-S) 和 Pan (α-BRD4-N) 抗体。



图 2.人类BRD4-L/S敲入结构示意图以及基因型和表型分析概要。
(一种) hBRD4 -L/S敲入 mo使用模型的示意图显示了 CreloxP依赖性重组,该重组触发了 CAGGS 启动子驱动的FLAG - Myc标记的BRD4 -L/S转基因表达,该转基因位于一个普遍存在的Rosa26基因座中或组织特异性方式。 (乙) 流程图详细说明了涉及转基因小鼠基因分型的实验方案,包括剪尾和裂解、PCR、琼脂糖凝胶电泳和小鼠选择,用于研究PyMT诱导的肿瘤生长,有或没有 Cre 介导的BRD4转基因表达和下游表型、蛋白质和组织学分析。缩写: MMTV,小鼠乳腺肿瘤病毒。

我们的基因分型程序的优势是独特的 PCR 协议,用于检测小鼠中hBRD4-L和hBRD4-S的存在,使用人类等位基因特异性引物扩增每个BRD4的共同或独特编码区域 异构体(图3A 为基因图,表 2 为引物信息)。特异于hBRD4-L外显子 13 和 15的 PCR引物对 (参见图 3A, hBRD4-L ,引物位置箭头)用于扩增hBRD4-L转基因, 它产生 216 bp 的 PCR 产物(图 3B, hBRD4-L凝胶)。为了扩增hBRD4-S ,我们使用与常见hBRD4外显子 11 退火的正向 PCR 引物, 和hBRD4-S外显子 12特有的反向引物(参见图 3A, hBRD4-S ),产生136 bp 的 PCR 产物(图 3B , hBRD4-S凝胶)。通过使用与FLAG序列退火的正向引物和识别hBRD4外显子 3(图3A, hBRD4-Pan )的反向引物,对任一 hBRD4转基因进行快速独立验证,产生520 bp的 PCR 产物 (图 3B, hBRD4-泛凝胶)。人类 BRD4转基因被插入位于小鼠 6 号染色体上的Rosa26基因座中。为了扩增该区域,我们选择了位于Rosa26基因座前病毒整合位点两侧的正向和反向引物对(图 3A;Soriano,1999)。检测到代表未插入基因座的 600-bp PCR 产物将表明没有BRD4转基因,或者如果同时检测到BRD4-L或-S转基因,则表明存在杂合构型。
为了检测hBRD4-L/S 表达所必需的Cre转基因的存在,使用了Cre特异性引物对(He等人,2003;Wu等人,2020),产生 374 bp 的 PCR 产物(图3B, MMTV-Cre凝胶)。使用这对引物,无法确定Cre的接合性;然而,对于 Cre 介导的loxP重组,纯合Cre基因型 ( Cre/Cre ) 不是必需的(参考文献 17)。有时, Cre 蛋白会在某些杂合MMTV-Cre小鼠品系(例如A 系)中引起意想不到的表型,例如泌乳缺陷(Yuan等人,2011),或某些 Cre 过表达小鼠的染色体异常,其中 DNA损伤在有丝分裂后的精子细胞中被诱导,这可能是由于哺乳动物基因组中loxP样序列触发的重组( Schmidt -Supprian和Rajewsky , 2007) 。因此,本方案中使用的所有 Cre 小鼠品系均来自 D 系(Wagner等人,1997),并且培育成仅携带一份用于组织特异性表达的Cre基因,以避免过度表达引起的缺陷。 由于单独的 hBRD4-L或hBRD4-S都不足以诱导小鼠乳腺肿瘤(我们未发表的数据),致癌PyMT 用于在雌性小鼠的乳腺脂肪垫中诱导肿瘤形成(Guy等,1992)。使用PyMT外显子 2 特异性正向和反向引物(图 3A),产生569 bp的 PCR产物(图 3B, MMTV- PyMT凝胶)。半合子雌性MMTV- PyMT 老鼠是从杰克逊实验室购买的,因为它们生产 用于乳腺癌研究的相当大的乳腺脂肪垫肿瘤,因此该方案中的所有小鼠都被培育成只携带一份MMTV- PyMT 基因。



图 3. 显示特定转基因和Rosa26基因座上的引物退火位点的基因图,以及预期 PCR 产物的大小。
(A) hBRD4 -L 、 hBRD4-S、MMTV-PyMT和MMTV-Cre转基因和Rosa26基因座的基因组图谱。每个 PCR 引物对的位置显示为每个基因图上方的行。 (B)琼脂糖凝胶图像显示每个引物对的特异性及其产生的 PCR 产物的大小。 通过 PCR 监测的转基因的存在 (+) 或不存在 (-) 显示在同一凝胶中不相邻泳道(由垂直虚线标记)的成对凝胶图像(相同曝光)中。 DNA 梯子(碱基对)标记在左侧。还指出了代表引物二聚化 (*) 的条带。

使用上述选择性 PCR 策略以及适当的育种方案,可以轻松进行hBRD4 -KI基因分型,以研究条件性 hBRD4同种型表达对癌症起始和肿瘤生长的影响。然后可以进行各种下游表型的分析,包括肿瘤大小/重量测量、形态苏木精和伊红 (H&E) 染色,以及通过西方印迹和免疫组织化学 (IHC) 检测异构体表达 (图 2B)。重点介绍了关键步骤,包括使用高盐蛋白提取缓冲液、湿转移和 BRD4 亚型特异性/泛抗体检测 BRD4 蛋白亚型(BRD4-L ~200 kDa和 BRD4-S ~120 kDa )在随附的蛋白质印迹程序中。与半干蛋白转移相比,我们的方案提供了始终如一的更好的质量和高分子量蛋白转移的可靠性,同时大大降低了背景。

关键字:BRD4 异构体, BET 溴结构域, 敲入转基因小鼠, 基因分型, PyMT, 乳腺癌, TNBC, 蛋白质印迹

材料和试剂


醋酸,冰醋酸,ACS 级(Fisher Scientific,目录号:A38C-212)
琼脂糖粉(Research Products International,目录号:A20090-500.0)
抑肽酶(Fisher Scientific,目录号:BP250310)
BRD4 抗体(内部;描述于 Wu等人,2006 和 2016 年)
α -Cre抗体(Millipore,目录号:69050-3)
直接 PCR 裂解反应(尾)溶液( Viagen ,目录号:102-T)
DNA Ladder,1 kb Plus(Thermo Scientific, GeneRuler ,目录号:SM1333)
干冰
耳标(National Band and Tag Company,目录号:1005-1P)
ECL 检测试剂:
West Femto化学发光底物(Thermo Scientific, SuperSignal TM ,目录号:PI34095)
ECL HRP 底物( Advansta , WesternBright ® ,目录号:K-12045)
印迹检测试剂(Millipore-Sigma,ECL TM ,目录号:GERPN2209)
溴化乙锭,分子生物学级(Sigma,目录号:E7637-5G)
乙醇,200 Proof( Pharmco ,目录号:111000200)
乙二胺四乙酸钠盐(EDTA,Research Products International,目录号:E57020-500.0)
过滤纸:
纯纤维素色谱纸(Fisher Scientific, Fisherbrand TM ,目录号:05-714-4)
厚印迹滤纸,预切,7.5 × 10 cm(Bio-Rad,目录号:1703932)
凝胶上样染料,紫色,6 × (New England BioLabs Inc.,目录号:B7024S)
甘氨酸(Fisher Scientific,目录号:BP381-5)
异丙醇(2-丙醇,Fisher Scientific,目录号:A416-4)
标签胶带,自粘(Fisher Scientific,目录号:1590110R)
亮肽素(Fisher Scientific,目录号:50-114-6410)
液氮
甲醇( Pharmco ,目录号:339000000)
小鼠(见表 1)


表 1. 本协议中使用的小鼠品系。


 


微量离心管,1.5 mL,高压灭菌(USA Scientific,目录号:1415-2500)
硝酸纤维素膜,支撑,0.2 mm(Fisher Scientific, Cytiva 阿默舍姆_ Protran TM ,目录号:45-004-017)
NP-40(Thomas Scientific,目录号:C953H73)
PCR 引物 (Sigma-Aldrich)


 


表 2. 用于鼠尾 PCR 反应的引物。 
指出了数据库、核苷酸序列和预期的 PCR 大小。 


 
PCR 管,圆顶带盖,0.2 mL,8 条(Phenix Research Products,目录号:MPX-445)
胃蛋白酶抑制剂A(Fisher Scientific,目录号:501146412)
苯甲基磺酰氟(PMSF, Sigma,目录号:P7626)
磷酸酶抑制剂混合物2(Sigma,目录号:P5726)
磷酸盐缓冲液 (PBS), pH 7.4
塑料食品包装(Kirkland Signature,目录号:208721)
预染蛋白标准品:
双色标准(Bio-Rad,Precision Plus Protein TM ,目录号:1610374)
Duo Pre-Stained Protein Ladder(Li-Cor Biosciences Inc.,Chameleon ® ,目录号:92860000)
Multicolor Broad Range Protein Ladder( ThermoFisher , Spectra TM ,目录号:26634)
蛋白质测定染料浓缩物(Bio-Rad,目录号:500-0006)
蛋白质凝胶,4–20%,15 孔,15 µL(Bio-Rad,Mini-PROTEAN ® TGX Stain- Free TM ,目录号:4568096 )
与辣根过氧化物酶 (HRP) 或荧光偶联的抗兔抗体:
山羊抗兔IgG二抗(Southern Biotechnology,目录号:OB4050-05)
抗兔IgG,800 4 × PEG缀合物(Cell Signaling Technology, DyLight TM ,目录号:5151)
Reactive Brown 10,实用级(Sigma-Aldrich,目录号:R0385-25G)
脱脂奶粉(雀巢公司、康乃馨® )
氯化钠(NaCl,Fisher Scientific,目录号:BP358-10)
脱氧胆酸钠(Fisher Scientific,目录号:BP349-100)
十二烷基硫酸钠(SDS,Fisher Scientific,目录号:BP166-500)
氢氧化钠颗粒,ACS级(Fisher Scientific,目录号:S318-3)
锡箔(Reynolds Wrap,目录号:2866C)
组织均质管,2 mL( Bertin Corp., CKMix ,目录号:P000918LYSK0A.0)
Tris Base(Research Products International,目录号:T60040-5000.0)
α -β-微管蛋白(H-235)抗体(Santa Cruz Biotechnology,目录号:sc-9104)
Tween 20(Research Products International,目录号:P20370)
Universal SYBR® Green Supermix ,2 × (Bio-Rad, SsoAdvanced TM ,目录号:1725274 )
水 (Milli-Q ® )
水,无核酸酶( Ambion ,目录号:AM9937)
X 射线胶片(Fisher Scientific, Cytiva 阿默舍姆_ Hyperfilm TM ECL,目录号:45-001-508)
1 × SDS-PAGE 缓冲液(见配方)
1 ×湿转移缓冲液(见配方)
20 × TBS-T 缓冲液(见配方)
EDTA,0.5 M,pH 8.0(见配方)
乙醇,70%(见食谱)
溴化乙锭溶液,5 mg/mL(见配方)
mRIPA缓冲液,400 mM(见配方)
PCR 引物工作溶液,5 µM(参见配方)
蛋白酶 K 溶液,20 mg/mL(参见食谱)
Tris-Acetate-EDTA 缓冲液,1 × ,pH 8.3(TAE,参见配方)




设备


AC 成像系统 (Ultra-Violet Products Ltd., BioSpectrum TM )
分析天平(Mettler Toledo,目录号:PG5002-S)
离心机(Beckman Coulter, Allegra TM X-22R,目录号:392187)


 


离心机(Eppendorf,5424,目录号:EP-022620401)


 


耳标涂抹器,不锈钢(Braintree Scientific,目录号:EP1005S1)
电极电源线,黑色和红色(Bio-Rad,Sub- Cell TM GT)
冰柜,-20°C 和 -80°C
凝胶梳,15 孔,固定高度,0.75 厚度(Bio-Rad,Sub- Cell TM GT,目录号:1704445)
凝胶电泳槽(Bio-Rad,DNA Sub- Cell TM )
荧光或化学发光凝胶成像系统:
Odyssey 红外成像系统 (Li-Cor Biosciences Inc., 9120)
桌面胶片处理器(柯尼卡美能达,型号:SRX-101A)
Gel Trey,UV-透明,15 × 25 cm(Bio-Rad,Sub- Cell TM GT,目录号:1704419)
均质器( Bertin Corp.,PreCellys ® Evolution,目录号:P000062-PEVO0-A)
磁力搅拌棒,八角形(The Lab Depot,SpinBar ® ,目录号:58948-171-EA)
带热板的磁力搅拌器(Fisher Scientific, IsoTemp TM ,目录号:11-100-49SH)
微波(肯莫尔)
鼠标解剖工具
pH 计(Fisher Scientific, Fisherbrand TM Accumet Basic 和BioBasic TM ,目录号:13-636-AB15)
电源(Bio-Rad、 PowerPac 200)
冰箱,4°C
滚筒(Bio-Rad,目录号:1651279)
Sonic Dismembrator (Fisher Scientific, FisherBrand TM ,目录号:FB120)
用于迷你预制凝胶的 Tetra 垂直电泳槽(Bio-Rad,Mini-PROTEAN ® ,目录号:1658005)
热循环仪(Bio-Rad,T100 TM热循环仪)
镊子(Fisher Scientific, Fisherbrand TM ,目录号:12-000-123)
Vortexer(Scientific Industries Inc.,Vortex-Genie 2 – 6560,目录号:SI-0236)
水浴(Fisher Scientific, IsoTemp 220 TM ,目录号:15-462-20)
湿蛋白转移仪(Bio-Rad,Mini Trans-Blot Module,目录号:1703935)




软件


Adobe Photoshop 2020(版本 21.2.4)
ImageJ(1.53 版)


 


Li-Cor 图像工作室(5.2 版)
Microsoft Office 专业增强版 (Excel)
Microsoft Office 专业增强版 (PowerPoint)
UVP VisionWorksLS图像采集和分析软件(7.0.1 版)




程序


第一部分:基因分型程序
 


小鼠尾组织采集 - 第 0 天


具备基本水平的鼠标操作和饲养能力(例如,擦伤、处理和约束、性别鉴定、鼠标耳标等)。
根据您所在机构伦理委员会关于动物实验的规定,在出生后 2-3 周左右开始对小鼠进行基因分型。


 


图 4。 由于物理连接的 K14-Agouti盒, MMTV-Cre 小鼠具有浅黄色毛皮颜色,该盒编码 131-aa 蛋白调节褐黑素(黄色色素)产生(Hustad,1995)。


决定对哪些小鼠进行基因分型。注意:在此过程中提到的所有基因都观察到典型的孟德尔遗传。因此,如果父母的基因型已知,则使用Punnett 方格来预测每个育种对的任何潜在后代的基因型。不是来自基因纯合的父母的小鼠通常需要进行基因分型,除了MMTV-Cre后代,由于K14-Agouti ,当与 B6-WT 背景中的小鼠杂交时表现出“黄色”皮毛与MMTV-Cre基因共同遗传的转基因 (Yuan et al. , 2011)。因此,这些小鼠可以仅根据其皮毛的颜色进行视觉选择(图 4) 。


 


图 5. Mo使用尾部组织采集。
(A)微型剪刀、镊子和耳标涂抹器。 (B)止血粉。 (C)标记的 PCR 管。 (D)带有耳标的约束和磨损鼠标。 (E)典型鼠标尾巴的长度。 (F)老鼠尾巴剪断。 (G) 2 毫米小鼠尾巴组织样本的长度。 (H)用止血粉覆盖的老鼠尾巴。 (I)收获的小鼠尾尖样本。


标记小鼠,如果尚未标记(图 5A 和 5D)。
用每个相应鼠标的标签号标记一组 PCR 管(图 5C)。
约束和磨损鼠标(图 5D)。
使用锋利的手术剪刀,剪断大约 2 毫米的尾巴(图 5A、5E-5G)。


 


在小鼠尾巴的末端涂抹止血粉以减少出血(图 5B 和 5H)。
将鼠标放回笼子。
使用镊子将剪尾直接放入相应的 PCR 管中(图 5I)。
用异丙醇对手术剪刀进行消毒。
对所有需要进行基因分型的小鼠重复上述步骤。
如果不立即裂解,请将小鼠尾巴样本置于 4°C;如果超过一天,请将它们在 -20°C 下储存长达六个月(避免多次冻融循环)。


准备 Lys是反应工作溶液 - 第 1 天


每个 0.2 mL PCR 管需要70 µL含有 0.4 mg/mL 蛋白酶 K(见配方)的 Direct PCR Lysis Reaction (Tail) 溶液(见下面的步骤 C1) 。
所有尾样本所需的直接 PCR 裂解反应溶液 (Lysis Rxn Sol.)的总量。
例如,70 µL × 8个样品 = 560 µL 直接 PCR 裂解反应溶液


 


每 1 mL Direct PCR Lysis Reaction 溶液,需要 20 µL 蛋白酶 K 溶液。根据直接 PCR 裂解反应溶液的总量计算所需的蛋白酶 K 溶液的总量。 


 


例如., (560 µL Lysis Rxn Sol.)((1 mL Lysis Rxn Sol.)/(1000 µL Lysis Rxn Sol.))((20 µL Proteinase K Sol.)/(1 mL Lysis Rxn Sol.))=11.2 µL of Proteinase K Sol. 
将上述计算的蛋白酶 K 溶液总量添加到 Direct PCR Lysis Reaction 溶液的总量中,以制备 Lysis Reaction Working solution 。
例如,560 µL Lysis Reaction Sol。 + 11.2 µL 蛋白酶 K Sol。 = 571.2 µL 裂解反应工作溶液。


裂解尾尖组织——第 1 天


将大约 70 μL 的裂解反应工作溶液添加到每个剪断的小鼠尾巴中。
将热循环仪中的每个样品在 55°C 下孵育 12 小时(步骤 1),然后在 85°C 下孵育 45 分钟(步骤 2),然后保持在 4°C 或 12°C(取决于特定的 PCR 机器,步骤 3)。


 




处理粗组织裂解物——第 2 天


将样品从热循环仪中取出,并通过以 123 × g离心1 分钟来沉淀任何剩余的碎片。
用每只鼠标的标签号标记一组新的 1.5 mL 微量离心管。
将粗裂解物转移到标记的微量离心管中。


 


如果不立即使用,将裂解物在 4°C 下最多保存一周,或在 -20°C 下最多保存六个月。
 


引物选择——第 2 天


确定对每只小鼠进行基因分型所需的 PCR 样本总数。
需要对哪些小鼠进行基因分型,请参阅A2 节。这些标准还将规定哪些基因需要通过 PCR 确认。
一般来说,不是来自纯合基因的父母的小鼠需要进行基因分型以确定该基因的存在;有“黄色”皮毛的老鼠例外,表明存在MMTV-Cre 基因(见图 4 ;Yuan等人,2011)。
为了正确确定BRD4-L和BRD4-S转基因的接合性(如果父母双方都不是hBRD4同种型的纯合子,并且后代可能是BRD4转基因等位基因的杂合子),使用BRD4-L或-S引物对用于确认敲入,然后使用Rosa26引物对通过另一次 PCR 进一步验证。 Rosa26 PCR结果解读如下:
对于具有纯合hBRD4-L/L 的小鼠, hBRD4-S/S,或杂合hBRD4-L/S ,当使用Rosa26引物对时,琼脂糖凝胶中不会出现Rosa26 DNA 条带(图 3B, Rosa26凝胶)。
对于没有BRD4- KI 的小鼠或具有一个BRD4转基因副本的杂合Rosa26小鼠,当使用Rosa26引物对时, Rosa26 PCR 带将出现在琼脂糖凝胶中(图 3B, Rosa26凝胶)。
可选的BRD4-Pan引物对可用于确认/排除任一 BRD4转基因的存在,以及Rosa26引物对(图 3B, hBRD4 - Pan凝胶)。
使用MMTV- PyMT引物对(参考文献 18)的PCR始终包括在内,以确认MMTV- PyMT基因的存在,因为我们所有的PyMT育种者都只携带一份MMTV- PyMT 基因(图 3B, MMTV - PyMT凝胶)。
每个 PCR 引物对都需要包含具有已知基因型的阳性和阴性对照样品。


主混合溶液制备 - 第 2 天


所有引物的 Master Mix 溶液是 7.5 µL 2 × SYBR ® Green mix、1.5 µL 各自 PCR 引物工作溶液(浓度为 5 µM;参见配方)和 5 µL 无核酸酶水的混合物,用于每个 PCR管中总体积为 14 μL 的 Master Mix 溶液(表 3)。 


 

表 3. 样本小鼠基因分型矩阵说明了一种组织对几种不同类型的转基因小鼠进行基因分型所需的 PCR 管总数的方法。


 


在本例中,对 8 只转基因小鼠进行基因分型。绿色字体表示 PCR 管是可选的。


 


计算制备每种主混合液所需的每种试剂的量,如表 3所示。请记住要考虑增加大约10-30%的溶液体积以获得误差范围。
为了帮助组织和样本计算,在 Microsoft Excel 中设置了一个鼠标基因分型 Excel 电子表格,如表 4 所示, PCR Master Mix 计算如表 5 所示。



表 4.小鼠基因分型矩阵电子表格。


 



表 5.用于样本计算的 PCR Master Mix 电子表格。


 


试剂所需的量,并在推进前验证所有试剂的数量是否充足。
准备每个 Master Mix 解决方案。
矩阵中的相应管号标记一组新的 PCR 管(表 3) 。
对于每个底漆组,在每个 PCR 管底部添加 1 μL 的粗裂解液,然后添加 14 μL 的 Master Mix 溶液。这种添加顺序有助于区分添加的样本和未添加的样本。
对所有其他引物组重复相同的步骤。
将任何剩余的粗裂解物在 -20°C 下储存长达六个月。


 


在室温 (RT) 下,在 Allegra X-22R 离心机中以 123 × g离心样品 1 分钟。


聚合酶链式反应 (PCR) – 第 2 天


对于BRD4转基因检测,将 PCR 协议设置为 94°C 3 分钟(步骤 1)、94°C 10 秒(步骤 2)、60°C 30 秒(步骤 3)和 72°C 30 秒(第四步)。重复步骤 2-4 35 个循环。在 72°C 下完成方案序列 3 分钟,然后在 4–12°C 下保持。


 


BRD4转基因扩增的PCR 管加载到热循环仪中。
按 Run,当仪器提示时将 PCR 体积设置为 15 µL,然后按 OK。
步骤G1中所述设置的另一个热循环仪对其他样品执行 PCR ,但重复步骤 2-4 40个循环。
此 PCR 过程大约需要 1.5 小时才能完成。
如果不立即使用,请将完成的 PCR 样品在 4°C 下放置长达一周,或在 -20°C 下放置长达六个月。


 


准备琼脂糖凝胶溶液 - 第 3 天


称取约 3.0 g 琼脂糖,将其添加到 200 mL 的 1 × TAE 缓冲液(pH 8.3,参见配方)中,搅拌制成 1.5% 的琼脂糖溶液。


 


在微波炉中加热溶液约 3 分钟,或直到琼脂糖完全溶解。
在琼脂糖溶液中加入 15 – 20 µL 的 5 mg/mL溴化乙锭溶液(参见配方) 。
让溶液在室温下搅拌至微温。


凝胶铸造 - 第 3 天


使用胶带覆盖凝胶浇铸托盘的末端,确保密封外边缘。
将四个 15 孔梳子(总共 60 个孔)均匀地放入凝胶浇注托盘中。
将所有 1.5% 的凝胶溶液倒入凝胶浇注托盘中 - 足以使溶液达到距底部0.6 – 0.8 厘米的高度。
去除任何气泡并均匀分布液体。
凝胶大约需要 20-25 分钟才能凝固。


凝胶加载 - 第 3 天


从凝胶浇铸托盘的每一侧取下胶带,并将托盘放入电泳槽中。
取下凝胶浇铸梳并将凝胶浸入 1 × TAE 缓冲液 (pH 8.3) 中。如果不立即使用,请将凝胶板放入 1 × TAE 缓冲液中(参见配方),并在 4°C 下储存。
冰箱中取出 PCR 样品,在每个样品中加入 3 μL 的 6 ×凝胶上样染料。
分组加载样本,每个样本在开始泳道中使用 1 kb Plus DNA 梯,在每个 PCR 引物组的最后两个泳道中使用阳性和阴性对照。在每个引物组后跳过一口井并开始加载以下一组样品。通常, 8 µL DNA ladder 和10 µL每个 PCR 样品足以在凝胶中产生显着条带;然而,这个数量可能会因产生的 PCR 产物的数量而异。
加载样品后,将黑色/阴极 (-) 和红色/阳极 (+) 电缆连接到电泳槽,并打开电源。凝胶中的电流从阴极流向阳极(黑红色)——与 DNA 样品通过凝胶迁移的方向相同。注意不要切换电极电源线。
将电压 (V) 设置为150 伏- 安培数 (A) 将变为2.00 A - 让凝胶运行 30-45 分钟,或直到上样缓冲液中的两种染料完全分离。


凝胶成像——第 3 天


将凝胶从凝胶浇注托盘中取出并将其浸入蒸馏水中。
打开BioSpectrum AC 图像系统(如果尚未打开)。
打开装置并用 70% 乙醇清洁玻璃(参见食谱)。
将凝胶放在凝胶扫描仪的表面并关上门。
启动VisionWorks程序并登录。
在“照明”选项卡下,将透照装置转到紫外光上。
按采集和预览。
在 Lighting 选项卡下,调整 Zoom 和 Focus 以锐化图像。
在相机选项卡下,调整曝光时间(分钟和秒)以更改图像的强度。
按捕获以获取图像。
在编辑选项卡下,旋转图像。
按对齐进一步调整角度,然后裁剪图像。
单击文件并另存为。
使用以下格式保存图像:DD MM YY, Initials, Mouse Genotyping Gel #。


凝胶处理——第 3 天


由于溴化乙锭的毒性,任何含有这种化学物质的凝胶都应丢弃在经过批准的废物容器中。


凝胶处理——第 3 天


使用 Adobe Photoshop


 


在 Adobe Photoshop 中打开扫描的图像。在图像面板下,单击图像旋转以正确定位图像。对于 180 度,单击 180°。对于特定角度,单击任意。
正确定位后,单击图像选项卡,然后单击模式;并单击灰度。
将出现一个带有忽略更改的弹出框,因此单击放弃。
在右侧窗格中,单击调整并选择反转。
通过单击亮度/对比度在调整面板下切换图像的对比度和亮度。
将图像另存为JPEG 或 TIFF 文件。
微软PowerPoint
将 Adobe Photoshop 中的黑白凝胶图像放入 PowerPoint 中,并用鼠标基因分型矩阵中的正确 PCR 管编号标记凝胶通道(表 3)。
它们对应的分子量标记 (M) 。


凝胶解读——第 4 天


请参阅图 3B作为正确波段识别的指南。
矩阵(表 3)中列出的每个 PCR 管的结果。
记录每只老鼠的基因型。


用于蛋白质印迹的肿瘤解剖和组织处理
肿瘤解剖


安乐死荷瘤小鼠(图 6A 和 6B)。收集额外的尾部组织用于备份基因分型,并储存在 -20°C。
在小鼠腹部顶部做一个 5 毫米的水平切口,使用微型剪刀刺穿小鼠的皮肤(图 6C)。
将切口下方的皮肤拉到尾部以暴露腹盆腔(图 6D)。
肿瘤将位于腹膜周围的脂肪垫组织中(图 6E)。
从乳腺脂肪垫 #4 和 #5 中取出肿瘤,注意不要带走任何周围组织(图 6F)。


 


现在将剩余的皮肤拉到颅骨主切口上方,直到露出整个胸腔(图 6G)。
肿瘤将位于附着在皮瓣后侧的脂肪垫组织中(图 6H、I 和 J)。
分别从乳腺脂肪垫 #3、#2 和 #1 中取出肿瘤,注意不要带走任何周围组织(图 6K)。


 


使用分析天平记录所有乳腺脂肪垫肿瘤的单个肿瘤重量和总肿瘤重量(参见图 6K)。
将每个肿瘤放入标记的微量离心管中(带有鼠标标签号、乳腺脂肪垫位置、日期和样本类型)。如果肿瘤太大而无法放入微量离心管中,请将其切成碎片并放入管中。
将样品微量离心管浸入液氮中以快速冷冻样品,并将其储存在-80°C。




 


图 6.小鼠乳腺PyMT肿瘤采集。
(A) CO 2安乐死系统。 (B)带有乳腺脂肪垫 #1–5 标记的安乐死小鼠。 (C)切口位于中线的鼠标。 (D)中线切口下方有皮瓣的小鼠从尾部拉出以露出腹膜。 (E)腹盆腔肿瘤。 (F)从乳腺脂肪垫 #4 和 #5收获PyMT肿瘤。 (G)中线切口上方有皮瓣的小鼠在颅骨上拉动以露出胸腔。 (H)鼠标 上部解剖右侧暴露以显示乳腺脂肪垫肿瘤#1-3。 (一)鼠标 上部解剖左侧暴露以显示乳腺脂肪垫肿瘤#1 和#2。 (J)鼠标 上部解剖最左侧暴露以显示乳腺脂肪垫肿瘤#2 和#3。 (K)收获的总乳腺脂肪垫肿瘤。缩写:MFP,乳腺脂肪垫; L, 左; R,对。


肿瘤处理


将冷冻样品从 -80°C 中取出,放入干冰中。
将冷冻组织转移到CKMix组织匀浆管 (2 mL ) 中,并添加mRIPA (400 mM) 缓冲液(参见配方-对正确分离 BRD4 与染色质很重要) 。


 


均质化肿瘤组织 PreCellys Evolution 组织均质器。
打开PreCellys Evolution 组织均质器。
在机器顶部装满干冰并盖上盖子。
转到主屏幕并选择软协议(具有以下规格:管体积 – 2 mL;旋转速度 – 5,800 rpm;循环 – 2 × 15 s;暂停 – 30 s;冷冻– 开启;温度 – 4°C;和模式 – 自动)、开始,然后按 OK。


 


确保样品完全均质化(即,液体中没有可见的大肿瘤块)。如果没有,请再次运行软协议。
完成后,将盖子打开一半,让干冰在冷却室中蒸发,然后关闭机器。
将样品放在冰上。
将样品转移到一组带标签的 1.5 mL 微量离心管中。从每个均质样品中取出尽可能多的液体。


 


在 20,000 × g和 4°C 下将样品离心 30 分钟。
离心后,将至少 50 μL 的上清液转移到第二组标记的管中。根据需要制作额外的 50 – 100 µL 等分试样。
为每个肿瘤样本保留一份样本以确定蛋白质浓度,将剩余的样本等分试样快速冷冻在液氮中,并将它们储存在 -80°C。
执行 Bradford 测定以测量每个样品等分试样的近似蛋白质浓度。为此,根据制造商的说明,从 Bio-Rad 蛋白质检测染料浓缩试剂 (5 × ) 中制备 1 ×溶液。


 


对于 Bradford 测定校准曲线,使用 1 × Bradford 染料制备 BSA 蛋白标准品。


蛋白质印迹程序
制备 25 – 50 µg蛋白质样品,来自任一细胞系(参见前面步骤 B9中的肿瘤处理部分注释)或已使用mRIPA (400 mM) 缓冲液均质化的PyMT肿瘤。对于正常组织,内源性 BRD4-S 检测需要约50 µg 蛋白质样品。
浇注 8% SDS-PAGE凝胶,或使用 4–20% 预制梯度凝胶。
加载 5–10 µL 的 5 mg/mL 蛋白质样品和预染色标准(通常为 5 µL,取决于公司来源)。
进行凝胶电泳。使用内部 8% 凝胶时,从 100V 开始,然后在预染色标准液进入分离凝胶后增加到200V 。按照制造商的说明运行预制凝胶。
×湿转移缓冲液(参见配方)冷却至 4°C(重要)。
使用 Bio-Rad Mini Trans-Blot Module进行湿转印:
组装蛋白质转移装置时,注意不要在 1 × Wet Transfer 缓冲液中产生气泡,因为这些气泡会干扰蛋白质转移并可能阻止蛋白质完全转移。
将传送带盒放在其透明的一侧。
一块薄湿海绵浸入冷的 1 × Wet Transfer 缓冲液中,然后将其放在卡带中间。
接下来,将一张厚滤纸浸入冷的 1 × Wet Transfer 缓冲液中,并将其放在海绵顶部,确保它们尽可能居中。根据需要切割。
使用滚筒去除滤纸上的任何气泡。
将一张浸入冷 1 × Wet Transfer 缓冲液中的硝酸纤维素膜放在滤纸上并去除任何气泡。
将 SDS-PAGE 凝胶放在硝酸纤维素膜的顶部并去除任何气泡。


 


再铺一层厚滤纸,浸入冷的 1 × Wet Transfer 缓冲液中,去除任何气泡。
最后,将一块浸入冷 1 × Wet Transfer 缓冲液中的薄海绵放在滤纸上,然后合上盒式夹层。
将盒式磁带放入设备中,使盒式磁带的黑色面朝向蛋白质转移装置的黑色面。
对任何后续凝胶重复上述步骤,然后将冰袋放在蛋白质转移装置的黑色一侧。
用任何剩余的冷 1 × Wet Transfer 缓冲液加满蛋白质转移装置,并将装置置于 4°C。
将盖子放在传输装置的顶部并将其连接到电源。
打开电源并将电压设置为 50 V,持续 720 分钟(重要)。
按下运行按钮开始湿蛋白转移。
 


完成后设备会自动关闭。
从 4°C 中取出转移装置,将每个膜置于用 1x TBS-T 缓冲液(见配方)在室温下稀释的 5% 脱脂牛奶溶液中 10 分钟。


 


计算每种抗体的量和稀释所需的稀释剂。一抗 BRD4 抗体:抗 BRD4-N(用于检测 BRD4-L 和 BRD4-S)和抗 BRD4-L 抗体的比例为 1:2,000,抗 BRD4-S 抗体的比例为 1:1,000(例如,将 3 μL 的抗 BRD4-N 抗体添加到 6 mL 的 5% 脱脂牛奶中)。
在 4°C 下轻轻摇动将膜在 BRD4 一抗中孵育过夜(或在室温下孵育 2 小时以检测更丰富的 BRD4-L 蛋白)。
过夜孵育后,将印迹从 4°C 中取出,倒出 5% 脱脂牛奶溶液,并用四次去离子水冲洗轻轻去除任何残留的牛奶溶液。
印迹中倒出水,并用 8-16 mL 1 × TBS-T 缓冲液替换(参见配方;确切体积取决于所用腔室/托盘的大小),使膜在液体中自由漂浮.
让印迹在 RT 处晃动 5 分钟。
× TBS-T缓冲液清洗所有印迹,共清洗 3 次。
将印迹放回1 × TBS-T 溶液中的 5%脱脂牛奶中,并以 1:10,000–4,000 稀释度添加二抗(例如,在 6 mL 5% 脱脂牛奶中,添加 0.6 µL 二抗1:10,000 稀释,1.5 µL 二抗用于 1:4,000 稀释)。
在 RT 的轨道摇床上轻轻旋转孵育二级抗体 1 小时。
倒出牛奶,用去离子水清洗四次,然后用四次 1 × TBS-T 冲洗,中间孵育 5 分钟,如步骤 10-13 所述。
在最后一次 TBS-T 洗涤后,用 1 × PBS(pH 7.4 )替换溶液,因为该溶液与荧光信号更兼容(例如,Li-Cor Odyssey 成像系统)。或者,如果使用由柯尼卡桌面胶片处理器显影的 X 射线胶片进行化学发光检测,则添加 ECL 检测试剂。


 


BRD4 蛋白异构体检测的代表性图像见图 7。


 


图 7.Western 印迹显示在来自三个代表性 Cre 表达小鼠的三个 PyMT 肿瘤中条件诱导的人 BRD4-S 过表达。
来自三只没有 Cre 表达的小鼠的 PyMT 肿瘤被包括在内作为对照。蛋白质大小标记(以 kDa 为单位)显示在每个凝胶图像的左侧。




食谱


5 µM PCR 引物工作解决方案
要为每个 Master Mix 溶液制备 PCR 引物工作溶液,如表 3所示,在 180 µL 核酸酶中加入 10 µL 正向 PCR 引物 ( 100 µM)到 10 µL 反向 PCR 引物 ( 100 µM) - 无水,用于每个基因特异性引物对。


0.5 M EDTA (pH 8.0)
对于 1 L 溶液,将 186.1 g EDTA 添加到 800 mL Milli-Q 水中。在磁力搅拌器上剧烈搅拌,并用 NaOH 颗粒将 pH 值调节至 8.0。用 Milli-Q 水将所得溶液调至 1 L。


 


1 × Tris-Acetate-EDTA (TA E, pH 8.3)缓冲液
对于 20 L 溶液,将 96.8 g Tris 碱、22.84 mL 冰醋酸和 40 mL 0.5 M EDTA (pH 8.0) 加入足量的去离子水中,使最终体积达到 20 L。
对于 1 L 溶液,将 4.84 g Tris 碱、1.14 mL 冰醋酸和 2.0 mL 0.5 M EDTA (pH 8.0) 添加到足够的去离子水中,最终体积为1 L。


5 mg/mL 溴化乙锭
在 200 mL 的高压灭菌/去离子水中加入 1 g 溴化乙锭。将溶液放入锡箔包裹的玻璃容器中以避免光降解,并在 4°C 下储存。


 


70% 乙醇
对于 1-L 溶液,将 700 mL 乙醇添加到 300 mL Milli-Q 水中。


蛋白酶 K 溶液 (20 mg/mL)
要制备 50 mL 的蛋白酶 K 缓冲液,请将 20 mL 的甘油、0.5 mL 的 1 M Tris(pH 7.5)和 50 μL 的 1 M 氯化钙添加到足够的去离子水中,最终体积为 50 mL。将100 mg蛋白酶 K 粉末(Fisher Scientific,目录号:BP1700-100)溶解在 5 mL 蛋白酶 K 缓冲液中,并将所得溶液以1-mL 等分试样储存在 - 20°C 。


mRIPA (400 mM) 缓冲液
对于 50 mL 的mRIPA (400 mM)溶液,将 2.5 mL 的 1 M Tris-HCl(pH 7.5,最终 50 mM)、4 mL 的 5 M NaCl(最终 0.4 M)、5 mL 的 10% NP 混合在一起-40(最终 1%)、1.25 mL 10% 脱氧胆酸钠(最终 0.25%)、0.1 mL 0.5 M EDTA(最终 1 mM)和蛋白酶抑制剂(由 0.5 µM PMSF、50 µg/mL 亮肽素组成、50 µg/mL Aprotinin、1 µg/mL Pepstatin A 和 500倍稀释的 Sigma 磷酸酶抑制剂混合物 2)。


1 × SDS-PAGE 缓冲液
对于 20 L 溶液,将 60 g Tris 碱、288 g 甘氨酸和 20 g 十二烷基硫酸钠 (SDS) 添加到足够的去离子水中,使最终体积达到 20 L。
对于 1 升溶液,将 3 克 Tris 碱、14.4 克甘氨酸和 1 克 SDS 加入足量的去离子水中,使最终体积达到 1 升。


1 ×湿转印缓冲液
对于 1 L 溶液,将 25 mL 的 1M Tris-HCl (pH 8.0)、14.41 g 甘氨酸、1 g SDS 和 200 mL 甲醇加入足够的去离子水中,最终体积为 1 L。


20 × TBS-T 缓冲液
对于 1 L 溶液,将 200 mL 的 1 M Tris-HCl (pH 8.0)、175.32 g 的 NaCl 和 10 mL 的 100% Tween 20 添加到足够的去离子水中,使最终体积达到 1 L。




致谢


我们感谢Chien -Fei Lee博士在协议开发过程中提供的技术帮助和指导,感谢Claire Chiang 和 William Chiang 对手稿的评论。此处详述的协议主要从Wu等人描述的程序扩展而来。 (2020 年)。这项工作得到了 NIH 赠款1RO1CA251698-01和 CPRIT 赠款RP190077的部分支持。




利益争夺


作者没有经济利益冲突。
伦理


随意获取食物和水。所有实验均按照 UTSW 机构动物护理和使用委员会 (IACUC) 批准的协议 #2017-102140 进行。




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引用:Lewis, M. P., Wu, S. Y. and Chiang, C. M. (2022). Conditional Human BRD4 Knock-In Transgenic Mouse Genotyping and Protein Isoform Detection. Bio-protocol 12(7): e4374. DOI: 10.21769/BioProtoc.4374.
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