Zebrafish Science Monitor Vol 4(1)

June 10, 1996

PREPARATION OF DNA-NLS COMPLEXES FOR MICROINJECTION INTO FERTILIZED ZEBRAFISH EGGS

By P. Collas and P. Aleström, Department of Biochemistry, Norwegian College of Veterinary Medicine, PO Box 8146 Dep., N-0033 Oslo, Norway

Introduction

We have previously reported the binding of the nuclear localization signal (NLS) of SV40 T antigen to plasmid DNA, and the induction of transgene expression following injection of DNA-NLS complexes into the yolk of fertilized zebrafish eggs (Collas et al., 1996). NLS peptides were bound to DNA by simple ionic interaction at a 100:1 molar ratio of NLS:DNA. NLS peptides bound to plasmid DNA efficiently target DNA-NLS complexes to nuclei. Transient reporter gene expression with 104 plasmid copies injected per egg using this system is similar to that with 106-107 copies injected without the NLS peptide (see, e.g., Stuart et al., 1988; Culp et al. 1991; Collas et al. 1996). Binding NLS peptides to 106 plasmid copies or more is detrimental to transgene expression and zebrafish embryo survival (Collas et al., 1996).

We present here the procedure used in our laboratory to prepare plasmid DNA-NLS complexes for cytoplasmic injection into zebrafish eggs. Complexes are prepared at a ratio of 100 moles of NLS per mole of DNA. It is assumed that 10,000 plasmid copies are injected per egg, in a volume of 250 pl. Note that plasmid DNA can be in supercoiled or linear form. The recipe is for the following NLS peptide: CGGPKKKRKVG-NH2 (Collas et al., 1996a).

Procedure

1. Calculate the intended concentration of plasmid DNA in the injection solution, taking into account DNA copy number and volume to be injected per egg. Prepare a DNA solution at 100x this concentration in H₂O.

2. Calculate the number of moles of NLS peptide required to obtain a final NLS:DNA molar ratio of 100:1. Calculate the corresponding number of grams of NLS (M = 974), and prepare a 5x NLS solution in H₂O (from an initial frozen stock dissolved in ddH₂O at 1 mg/ml).

3. Prepare 1 ml of 1 M KCl in H₂O, and 1 ml of 1% phenol red in H₂O.

4. Prepare the injection solution by mixing together:

Sterile H₂O		68 ul
1 M KCl		50 ul
1% Phenol red		40 ul
DNA solution		2 ul
NLS solution		40 ul

TOTAL		200 ul

and incubate for 30 min at room temp.

5. Filter the solution and proceed with injections (Collas et al., 1996b). This solution can be stored for at least two weeks at 4^oC.

References

Collas, P., H. Husebye, and P. Aleström (1996a) The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei. Transgen. Res. 5:in press.

Collas, P., H. Husebye, and P. Aleström (1996b) Transferring foreign genes into zebrafish eggs by microinjection. In Transgenic Animals - Generation and Use, L.M. Houdebine, ed., Amsterdam: Harwood Academy Publishers, in press.

Culp, P., C. Nüsslein-Volhard, and N. Hopkins (1991) High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs. Proc. Natl. Acad. Sci. USA 88:7953-7957.

Stuart, G.W., J.V. McMurray, and M. Westerfield (1988) Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103:403-412.

The Zebrafish Science Monitor, Vol 4(1)

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