Protocols

Table of Contents

Cloning your Transgenic Vector                                                                                       

Preparing DNA vector                                                                                                    

Transformation of competent bacteria                                                                           

Cloning your vector                                                                                                        

Designing oligonucleotide linkers                                                                                   

Annealing linkers                                                                                                            

Ligation of linkers to cDNA                                                                                             

Restriction analysis of your vector                                                                                

Sequencing your vector                                                                                                

Generating transgenic or targeted ES cells                                                                     

Electroporation                                                                                                               

Drug selection of ES cells                                                                                              

Picking colonies                                                                                                              

Replica plating 96-well plates                                                                                        

Freezing 96-well plates of colonies                                                                               

Thawing cells from 96-well plates                                                                                 

LacZ staining of ES cells                                                                                               

Human alkaline phosphatase (hPLAP) staining of ES cells                                            

eGFP visualization of ES cells                                                                                        

ES cell DNA extraction and purification from 96-well plates                                         

ES cell DNA extraction and purification from 24-well plates                                         

Southern hybridization                                                                                                   

Cre excision to test cDNA and second reporter expression                                         

Genotyping mice                                                                                                               

LacZ staining                                                                                                                 

Human alkaline phosphatase (hPLAP) staining                                                              

eGFP                                                                                                                              

Staining tissue sections                                                                                                    

LacZ tissue staining                                                                                                       

Human alkaline phosphatase (hPLAP) staining                                                              

eGFP                                                                                                                              

Sorting eGFP positive cells                                                                                              

 

Cloning your Transgenic Vector

Preparing DNA vector

Your DNA vector is shipped on Whatman paper. To derive the DNA:

Transformation of competent bacteria

Cloning your vector

Multiple methods exist for subcloning a cDNA of interest into expression vectors. You may require oligonucleotide linkers to generate compatible restriction sites between the cDNA and the transgenic vector.

Designing oligonucleotide linkers

G A A T T C C T C G A G G A A T T C

C T T A A G G A G C T C C T T A A G

        EcoRI            KpnI              EcoRI

 

For example, if an EcoRI site is at the 3’ end of the cDNA, and you want to subclone into the KpnI site of the transgenic vector, the linker would look like the red and blue portion of the above oligonucleotide. The black portion represents the 3’ end of the cDNA. Therefore you would order a 12-mer that is 5’-AATTCCTCGAGG-3’.

Note that in cloning a cDNA into iZAP or iZEG, you do not need to put the cDNA in-frame with the Alk Phos or EGFP, because the Alk Phos and EGFP are preceded by an IRES sequence.

Annealing linkers

Ligation of linkers to cDNA

Restriction analysis of your vector

Restriction enzyme digests may be used to confirm the insertion and orientation of the cDNA insert in the transgenic vector.

Sequencing your vector

The vector can be sequenced to confirm the position of your cDNA relative to the expression of the reporter gene.

Generating transgenic or targeted ES cells

The transgenic vector is ideally introduced into murine embryonic stem (ES) cells through electroporation or lipofectin transfection methods. We recommend using ES cells rather than pronuclear injection to generate transgenic mice, because you can select ES cell clones with strong lacZ expression and a single-copy transgene. This provides widespread expression in the mice you generate and avoids recombination between multiple tandem loxP sites. Remember to use feeder cells that are neomycin- or puromycin-resistant, depending on which drug you are selecting with.

Electroporation

Day 0: Prepare plates of freshly inactivated (mitomycin C-treated) feeders in PMEF medium.

Day 1: Thaw one vial of ES cells (an equivalent of one fifth of a 10 cm plate) onto two 10 cm plates with a confluent lawn of feeders in ES cell medium. Feeder plates can be used for several days after being prepared.

Day 2: Refeed ES cells (with cold medium). The cells should be well-defined colonies with clear edges.

Day 3: Passage cells 1:5 or 1:7 into 10 cm plates.

Day 4: Refeed ES cells. Prepare DNA for electroporation by linearizing the vector.

Day 5: Electroporate ES cells:

Day 6: Refeed ES cells with normal medium. There should be ~50% cell death observed.

Drug selection of ES cells

Day 7: Drug selection with either G418 or puromycin should begin 48 hour after electroporation or transfection. Use the drug concentration pre-determined by a dose response curve.

Day 8: Refeed the cells with freshly added drug.

Day 9: Refeed. There should be obvious cell death. Drug-sensitive colonies have edges that become less sharp, and cells that are dead on top. Drug-resistant colonies have clear edges and look shiny.

Day 11: There should be massive cell death. Refeed with medium containing drug.

Day 12: There will be some colonies that continue to die. Resistant colonies should be more visible as round, nut-shaped colonies. The edges of colonies should be sharp, with no brownish centre. Colony sizes may vary.

Day 13: Some residual cell death may be observed. Two types of resistant colonies should be observed: fast growing colonies that are visible to the naked eye, and smaller colonies that grow 3-4 days slower than do the large ones. The fast growing colonies may have varying morphologies: tightly-packed colonies (OK), dense centred but with a peripheral rim of flattened cells (OK) which may be fairly large (OK), or completely flattened colonies (not suitable). The slower-growing colonies are likely resistant, but express less drug resistance genes. These colonies are not typically used for injection.

Day 14: Refeed cells.

Day 15: Refeed cells.

Picking colonies

To screen individual ES cell colonies for reporter expression, pick single colonies into individual wells of 96-well plates. By day 16, colonies should be large enough to be picked.

Day 17: Refeed the colonies early the next day.

Day 18: Refeed the picked colonies with fresh ES cell medium with drug.

Day 19: Refeed the picked colonies with fresh ES medium with drug.

Replica plating 96-well plates

Day 20: When ES cells in the 96-well plate become confluent, passage them 1:3.

Day 21: Feed cells with fresh medium.

Day 22: Refeed the cells. Place a Styrofoam box at -80°C in preparation for freezing the ES cells on feeders.

Freezing 96-well plates of colonies

Day 23: Two hours prior to freezing, change the media.

Thawing cells from 96-well plates

It is essential to rapidly thaw cells and get them into fresh ES medium.

LacZ staining of ES cells

Cells can be stained for lacZ expression one day after passage. The positive clones (typically 10% of total) can be passaged into two 24-well plates. One 24-well plate (gelatinized) can be used for DNA extraction, and the other 24-well plate (with feeders) is passaged into 6-well plates, then 10 cm plates. These cells are for frozen stocks.

X-gal staining solution: store unused solution wrapped in foil at -20°C. IMPORTANT: Start with PBS first, or else the other components will not dissolve.

Final concentration

Stock concentration

To make 10 mL

1X PBS

1X

9.35 mL

1 mg/mL X-gal in DMSO

40 mg/mL

250 mL

5 mM K3Fe(CN)6

500 mM

100 mL

5 mM K4Fe(CN)6

500 mM

100 mL

2 mM MgCl2

100 mM

200 mL

Human alkaline phosphatase (hPLAP) staining of ES cells

 

NBT/BCIP stain solution: make 20X in ddH2O, then dilute 1:20 and add NBT/BCIP.

20X concentration

2 M NaCl

0.2% sodium deoxycholate

0.4% NP-40

eGFP visualization of ES cells

eGFP expression can be assayed in live cells with a fluorescent microscope. 

ES cell DNA extraction and purification from 96-well plates

DNA from this procedure is used for Southern hybridization to check for single copy integration of the transgene. See below for extraction from 24-well plate (which is more reliable to work).

Lysis buffer. Make fresh before use.

Final concentration

Stock concentration

To make 10 mL

0.5% sarcosyl

10%

500 mL

200 mM NaCl

5 M

400 mL

10 mM EDTA (pH 8.0)

500 mM

200 mL

10 mM Tris-HCl (pH 8.0)

1 M

100 mL

1 mg/mL proteinase K

20 mg/mL

500 mL

ES cell DNA extraction and purification from 24-well plates

 

Lysis buffer. Make fresh before use.

Final concentration

Stock concentration

To make 10 mL

1% SDS

20%

500 mL

100 mM NaCl

5 M

200 mL

1 mM EDTA (pH 7.5)

500 mM

20 mL

10 mM Tris-HCl (pH 7.5)

1 M

100 mL

500 mg/mL proteinase K

20 mg/mL

250 mL

Southern hybridization

Day 1: Restriction enzyme digest

Digest 10 mg genomic DNA in a total volume of 40 mL. Digests should be performed overnight.

Day 2: Gel electrophoresis

Run your samples on a 0.8% agarose gel overnight, at 20-25V. Add the appropriate amount of loading dye.

Day 3: Southern transfer. Adapted from Manniatis, 9.44.

Day 4: Prehybridization and hybridization

Pre-hybridization solution. Store at -20°C. Use 0.2 mL solution/cm2 of Hybond N+ membrane.

Final concentration

Stock concentration

To make 25 mL

5X SSPE

20X

6.25 mL

5X Denhardt’s

50X

2.50 mL

1% SDS

10%

2.50 mL

0.1 mg/mL Denatured salmon sperm DNA

1 mg/mL

2.50 mL

50% Formamide

 

11.25 mL

 

20X SSPE

175.3 g NaCl

27.6 g NaH2PO4-H2O

7.4 g EDTA

Add 800 mL ddhH2O

Adjust the pH to 7.4 with ~6.5 mL 10 N NaOH

Make up the volume to 1 L

Autoclave

Hybridization solution. Store at -20°C.

Final concentration

Stock concentration

To make 25 mL

5X SSPE

20X

6.25 mL

1% SDS

10%

2.50 mL

0.1 mg/mL Denatured salmon sperm DNA

1 mg/mL

2.50 mL

10% Dextran sulphate

 

2.50 mL

50% Formamide

100%

11.25 mL

 

Probe preparation

Prepare probe according to the instructions in your kit.

Probe purification

Hybridization

Day 5: Wash

Cre excision to test cDNA and second reporter expression

Cre expression in lacZ positive ES cell clones can be used to confirm excision of lacZ and expression of the cDNA and the second reporter gene. This protocol is optimized for ES cells, and adapted from the protocol for LIPOFECTAMINE 2000 (Invitrogen).

Genotyping mice

Mice may be genotyped by Southern blot and probing for a fragment of the transgenic vector. Mouse earclips can be directly stained for b-galatosidase or alkaline phosphatase expression. eGFP can be directly visualized in earclips under a fluorescent microscope.

LacZ staining

Human alkaline phosphatase (hPLAP) staining

AP wash buffer

Stock concentration

To make 100 mL

1 M Tris-HCl pH 9.5

10 mL

5M NaCl

2 mL

1 M MgCl2

1 mL

ddH20

87 mL

 

NBT/BCIP stain solution: make 20X in ddH2O, then dilute 1:20 in NBT/BCIP.

20X concentration

2 M NaCl

0.2% sodium deoxycholate

0.4% NP-40

 

eGFP

eGFP fluorescence can be visualized in earclips and live animals using an appropriate light source and filter.

 

Staining tissue sections

LacZ tissue staining

LacZ fix solution

Stock concentration

To make 50 mL

25% glutaraldehyde

0.4 mL

250 mM EGTA pH7.3

1.0 mL

1 M MgCl2               

5.0 mL

PBS

43.5 mL

 

LacZ wash buffer

Stock concentration

To make 500 mL

1 M MgCl2

1.0 mL

1% sodium deoxycholate

5.0 mL

2% NP-40

5.0 mL

PBS

489 mL

 

LacZ stain buffer

Stock concentration

To make 75 mL

LacZ wash buffer

72.0 mL

25 mg/mL X-gal (in DMSO)

3.0 mL

K3Fe(CN)6

0.159 g

K4Fe(CN)6

0.123 g

Human alkaline phosphatase (hPLAP) staining

AP wash buffer

Stock concentration

To make 100 mL

1 M Tris-HCl pH 9.5

10 mL

5M NaCl

2 mL

1 M MgCl2

1 mL

ddH20

87 mL

 

NBT/BCIP stain solution: make 20X in ddH2O, then dilute 1:20 in NBT/BCIP.

20X concentration

2 M NaCl

0.2% sodium deoxycholate

0.4% NP-40

 

eGFP

eGFP can be detected in frozen sections. We have used anti-GFP, rabbit IgG fraction, HRP conjugate (Invitrogen A10260) primary antibody, Peroxidase goat anti-rabbit IgG secondary antibody (Vector Laboratories PI-1000), VECTASTAIN ABC Kit for goat IgG (Vector Laboratories PK-4005), and DAB Peroxidase Substrate Kit (Vector Laboratories SK-4100).

Sorting eGFP positive cells

Cells expressing eGFP may be sorted and collected using flow cytometry.