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Modified transgenesis protocol for X


Transgenesis protocol for X. laevis

(Hajime Ogino, 3/16/2004, modified 4/24/07)

 

Comments

We have modified the original protocol developed by Kroll and Amaya (Development 122, 3173-3183, 1996) to improve efficiency.  Consult our “previous version” of the transgenic protocol for general background and details of the method, published in Early Development of Xenopus laevis: A Laboratory Manual (Sive, H. L., Grainger, R. M. and Harland, R. M., Cold Spring Harbor Laboratory Press, 2000).  The protocol described here is the latest version of our protocol that includes both the modifications originally made for transgenesis in X. tropicalis (Offield, M. F. et al., Development 127, 1789-1797, 2000, Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002) and some further refinements.  The efficiency of transgenesis with this protocol is around 10%: ~30% of injected eggs cleave normally, ~70% of normal cleavers gastrulate normally, and ~50% of the remaining normally gastrulated embryos express a transgene.  The major changes from previous iterations of the protocol are as follows:

 

1) Use of oocyte extract instead of egg extract.  This modification was originally made for the transgenic procedure in X. tropicalis (Hirsh, N. et al. Dev Dyn. 225, 422-433, 2002), but also works well in X. laevis.

 

2) Use of MOH buffer instead of SDB for dilution of the sperm nuclei reactions (Offield, M. F. et al. Development 127, 1789-1797, 2000).

 

3) The restriction enzyme and MgCl2 may be omitted from the transgenic reaction mix.  While the use of restriction enzyme stimulates transgene integration, it can also damage the host genome.  Embryos carrying the damaged genome often develop abnormally and will show ectopic transgene expression.  If the transgene is shorter than 5 kb, the restriction enzyme and MgCl2 can be omitted from the reaction.  However, if the transgene is longer than 10 kb, the restriction enzyme and MgCl2 might be necessary to get consistent results.

 

4) 0.5% BSA is added to all solutions used after dejellying (i.e. 1x MMR for the post-dejellying rinse, the injection medium).  Following dejellying, the unfertilized eggs are easily activated by mechanical stimuli—the BSA prevents the egg activation that often happens during the post-dejellying rinse and egg loading into the injection dishes.  Omitting calcium from the dejellying and post-dejellying rinse solutions also helps to prevent the egg activation.

  In addition, the BSA appears to maintain egg activity for fertilization prior to injection and aid in the healing of injected eggs.  The underlying mechanisms are unclear, but the addition of BSA to the culture medium increases the number of transgenic embryos that gastrulate normally.

 

5) The plasmid backbones are fully removed from transgene constructs (Hirsh, N. et al., Dev Dyn. 225, 422-433, 2002).  In the original Kroll and Amaya protocols, plasmids carrying a transgene construct are linearized and used directly in the transgenic reaction.  However, we have found that the flanking vector sequence introduced with a transgene can interfere with correct expression of the transgene.  This appears to be enhancer/promoter-specific, but in severe cases, strong background expression can mask the “correct” expression pattern of the transgene Thus, we completely remove the plasmid backbone sequences from a transgene cassette by restriction enzyme digestion followed by gel electrophoresis.  The transgene is then purified from the agarose gel using the Qiaquick Gel Extraction kit (Qiagen), precipitated with ethanol once, and finally dissolved in 10 mM KCl when it is ready for use in transgenesis.


                           

  TOP: An embryo carrying a transgene in which the plasmid backbone has been left in the injected construct. 

  BOTTOM:  An embryo carrying the same transgene but without the plasmid sequence. 

 

STOCKS AND PREMADE BUFFERS

 

I. Stock Media

 

                                  

10 x MMR (with Ca2+) (1L) from stock solutions in grams
1 M NaCl 250 ml 4 M NaCl 58.44 g NaCl
20 mM KCl  20 ml 1 M KCl  1.49 g KCl
10 mM MgCl2 10 ml 1 M MgCl2  2.03 g MgCl2·6H2O
50 mM HEPES 50 ml 1 M HEPES 11.92 g HEPES
20 mM CaCl2 20 ml 1 M CaCl2  2.94 g CaCl2·2H2O

 

add water to 1 Liter, pH to 7.4-7.6 with NaOH

Autoclave if made from dry stocks. Otherwise just combine sterile solutions + sterile H2O in a sterile bottle.

 

10x MMR (No Ca2+)

Prepare as above, but omit 20 mM CaCl2.  This reagent is used in steps prior to injection to prevent egg activation.

10 x MBS SALTS (1 L) from sterile stock solns from grams
880 mM NaCl 220 ml 4 M NaCl 51.4 g NaCl
10 mM KCl 10 ml 1 M KCl 0.75 g KCl
10 mM MgSO4 10 ml 1 M MgSO4·7H2O 2.46 g MgSO4·7H2O
50 mM HEPES  50 ml 1 M HEPES  11.92 g HEPES
25 mM NaHCO3 25 ml 1 M NaHCO3  2.1 g NaHCO3

pH to 7.4-7.6 with NaOH, autoclave for sterilization

 

 

II. Sperm Nuclei Prep

 

·1.5 M Sucrose (Sigma S-0389)

                Dissolve 513.45 g in 1000 ml of deionized nanopure water, filter sterilize, and store in 11 ml aliquots at -20°C.

·1 M HEPES (Sigma H-8091)
                Dissolve 21.45 g in 90 ml deionized water. pH to 7.7, filter sterilize and store in 990 µl aliquots at -20°C.

·100 mM spermidine trihydrochloride (Sigma S-2501)

                Dissolve 0.255g in 10 ml of sterile water. Store in aliquots of 330 µl at -20°C.

·100 mM spermine tetrahydrochloride(Sigma S-1141)

                Dissolve 1.74g in 50 ml of sterile water. Store in 140 µl aliquots at -20°C.

·1 M dithiothreitol (DTT) (Sigma D-9779)

                Dissolve 1.54g in 10 ml of sterile water. Store in aliquots of 66 µl at -20°C.

·10% bovine serum albumin (BSA)(Sigma A-3294)

                Slowly dissolve 40 g by floating it on 375 ml of deionized water. It should slowly go into solution. Bring final volume to 400 ml and pH to 7.6 with KOH. Final solution should look yellowish. Store at -20°C in 4 ml aliquots.

·10 mg/ml leupeptin (Roche 1 017 128)

                Dissolve 10 mg in 1 ml of Dimethyl Sulfoxide (DMSO) (Sigma D-8418) and store in 14 µl aliquots at -20°C.

·0.3 M phenylmethylsulfonyl fluoride (PMSF) (Boehringer Mannheim 837 091)

                Dissolve 78 mg in 1.5 ml of ethanol and store in 14 µl aliquots at -20°C.

·0.5 M EDTA (J.T. Baker 8991-01)