Cas9 Genome Editing

The latest tool in genome editing – CRISPR/Cas9 – allows for specific genome disruption and replacement in a flexible and simple system resulting in high specificity and low cell toxicity. The CRISPR/Cas9 genome editing system requires the co-expression of a Cas9 protein with a guide RNA. With the protospacer-adjacent motif (PAM – the sequence NGG) present at the 3′ end, Cas9 will unwind the DNA duplex and cleave both strands upon recognition of a target sequence by the guide RNA. The functional cassette in the rescue donor vector can then be inserted into the unwound DNA. The repaired genome will now express your desired sequence with or without tags.

OriGene provides all the vectors and service for the Cas9-genome editing.  http://www.origene.com/CAS9/

pCas-Guide vector (SKU# GE100002) encodes Cas9 enzyme and the guide RNA. You can use OriGene free gRNA design tool to design your guide RNA. Have them synthesized by any commercial company such as IDT . Anneal them together to make a double strand DNA and ligate the dsDNA to pCas-Guide vector. OriGene also offers an annealing buffer (SKU# GE100007) for you to make the dsDNA.

pCas-Scramble (SKU#GE100003) contains a scrambled sequence in pCas-Guide vector. It is a good negative control that you can use for your work.

The gene specific rescue donor vector can be made through gene synthesis (www.blueheronbio.com) or from your own donor constructs. OriGene also provides donor vector construction service.  To order your donor vector service, please go to http://www.blueheronbio.com/Services/Genome-Editing.aspx

You can also select the following premade functional cassettes:

  • GFP-CMV-LoxP-Puro-LoxP
  • RFP-CMV-LoxP-BSD-LoxP
  • Luc-CMV-LoxP-Puro-LoxP

Cas9 Further Reading

IHC Antigen Retrieval

Antigen retrieval is necessary when you perform the IHC experiments on FFPE tissue. The most common Antigen retrieval method is Heat Induced Epitope Retrieval (HIER).  And there are three important factors to consider for successful HIER:

  • Optimal heating time
  • Good preparation of tissue section
  • Specific type of antigen retrieval solution

Optimal heating time: The heating time has to be optimized for each antigen and appliance.

Good preparation of tissue section: OriGene has more than 140,000 human tissue samples from U.S. medical centers using the industry’s most strict ethical requirements and IRB protocols. For the complete list of our tissue section, please check the following OriGene website: http://www.origene.com/Tissue/TissueSearch.aspx

Specific type of antigen retrieval solution: The main difference among different retrieval solutions is pH. Citrate solution is acidic, while EDTA solution is basic. Some antibodies may request an enzymatic antigen retrieval solution, like Trpsin and Pepsin solution (RTU) from OriGene (SKU# GB300016 and GB300017). You will have to optimize your solution to get the best staining result.  OriGene has all the solutions that you need for your IHC. You can find the complete product list from the following link.

http://www.origene.com/Anatomic_Pathology/IHC_Accessories.aspx

new transfection reagent for the multipotent stem cells

DreamFect™ Stem (TT100159 & TT100160) is an original, simple and efficient method especially developed to transfect multipotent stem cells with high transfection efficiency and superior transgene expression level.

1.       High transfection efficiency for multipotent Stem Cells

2.     Minimized toxicity due to reagent biodegradability and low DNA amount required

3.       Do not affect phenotype and differentiation potential

4.       Serum Compatible

5.      Simple, Ready-to-use and rapid (one vial, no specific buffer, no need magnetic plate)

http://www.ozbiosciences.com/dreamfect-stem-3.html

Do you need to linearize the plasmid DNA before stable transfection?

It is not necessary to linearize a plasmid before stable transfection. Linearize the plasmid will increase the chances of correct integration, but also reduces the transfection efficiency. So in the end, there is no big difference between these two methods.

Viral ORF Clones: Expression-ready for Viral Proteins

Over 2,000 TrueORF Clones are available for viral protein expressions

  • Covering over 120 human viruses
  • Codon optimized for human cell expression
  • All cloned in proven pCMV-Entry Vector
  • Myc-DDK tagged for convenient detection and purification
  • 60 destination vectors for additional applications

Transfection of Mouse N2a Cells using Lipofectamine/plus

1.  Quick thaw the cells in 37 C water bath and add to 10 ml of growth medium. Spin 5 min at 800 rpm to remove DMSO from freezing medium.  DMSO is toxic to growing cells.  Resuspend in 10 ml growth medium in 75 cm2 TC flask and place in 5% CO2/37 C incubator.

2. Plate approximately 5 x 105 cells into 60mm petri dishes. Wait until the cells reach 80% confluent.

3.  Dilute 2-4 ug DNA of each construct to be transfected into 250 ul of OptiMEM or DMEM medium. Add 8 ul of Plus Reagent (Invitrogen # 11514-015) to the DNA/MEM mix.  Let sit 15 min at room temperature.

4. Separately, mix 12 ul of Lipofectamine (Invitrogen # 18324-111) with 250 ul of OptiMEM or DMEM.

5. Mix DNA/MEM/Plus solution with Lipofectamine/MEM.  Let sit 15 min at room temperature.

6. Remove growth medium from 60mm cell cultures.  Rinse once with 2 ml OptiMEM or DMEM, replace with another 2 ml MEM to remove serum from the plate.

7. Add preincubated DNA/Plus/Lipofectamine/MEM solution to the cells.  Mix gently.

8. Grow cells 3 hr – overnight.  Add 2.5 ml N2a medium and continue culture.

If developing stable transfectants, add G418 (Gibco/Life Tech # 1181-031) to the growth medium  to select for positive cells.  Use at 400-800 ug/ml for selection of clones, and 200-400 ug/ml for maintenance.

9. 48 hours post transfection, harvest the cells for the future assay.

Growth medium:

DMEM, high glucose, w/L-gluatmine       Gibco # 11965-092     250ml

Fetal bovine serum, qualified           Gibco # 26140-079      50ml

OptiMEM 1 medium                    Gibco # 31985-070          250ml

Penicillin-Streptomycin            Gibco # 15140-122             5ml

Filter to 0.2 um.  Stable for several months at 4 C.

Immunohistochemistry

1. Solutions and reagents

1.1 Xylene

1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)

1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.

1.4. Distilled water (dH 2O)

1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0

To prepare Antigen Retrieval stock solutions:

- 10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dihydrate (C 6H 5Na 3O 7 2H 2O)
in 1 liter of dH2O. Add 5mL Tween-20.

- 1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0

1.6. 3% Hydrogen Peroxide

1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)

1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the secondary antibody)

1.9. Hematoxylin

1.10. Permanent Mounting Medium

2. IHC Protocol

2.1. Deparaffinization/Rehydration

2.1.1. Heat slides in an oven at 65 °C for 1 hour.

2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.

2.2. Antigen Retrieval

This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.

2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.

2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.

2.2.4. Let it cool down to room temperature (10 – 20 minutes).

2.2.5. Removes slides and rinse in TBST.

2.2.6. Proceed to Staining step.
2.3. Staining

2.3.1. Wash slides with TBST for 3 min on a shaker.

2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.

2.3.3. Wash slides three times with TBST (3 min each on a shaker).

2.3.4. Block slides with the blocking solution for 1 hour.

2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.

2.3.6. Apply primary antibody to each section and incubate overnight in the humidified
chamber (4 °C).

2.3.7. Wash slides three times with TBST (3 min each on a shaker).

2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer’s recommendation; incubate for 30 min at room temperature.

2.3.9. Wash slides three times with TBST (5 min each on a shaker).

2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).

2.3.11. Rinse sections with water.

2.3.12. Counterstain with Hematoxylin (generally 10 seconds).

2.3.13. Rinse sections with water.

2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by two rinses with Xylene (3 min each).

2.3.15. Mount coverslips on slides using permanent mounting medium