Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster.
Here we describe how to generate customized microinjection needles from glass capillary tubes. Controls demonstrate the range of variables and effects on needle tip shape using a standard Flaming/Brown micropipet needle puller. Needles generated with two-cycle pulls provide a wider range of needle shapes in a predictable fashion. We used the needle puller's ramp function for multiple-cycle programs to determine the useful range of heat settings inherent to the glass capillary tube. This articlefocuses primarily on the preparation of injection needles utilized for P-element-mediated germ-line transformation in Drosophila melanogaster that do not require the dechorionation of the egg. However, these types of needles can be usefulfor numerous other types of injections, such as RNA interference, homologous recombination mutagenesis, morpholinos, transient gene regulation, drug delivery, and the transfer of cytoplasmic factors that are useful in a wide range of biological systems ranging from plants to vertebrates. Using our standard needle, we correlate the survival of injected D. melanogaster embryos with transformation efficiencies and plasmid construct characteristics.
Miller
David F B
DF
Indiana University, Bloomington 47405, USA.
Holtzman
Stacy L
SL
Kaufman
Thomas C
TC
D000399:Air Pressure; D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics*; D004867:Equipment Design; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D005854:Germ Cells; D005898:Glass; D006358:Hot Temperature; D008845:Microinjections / Q000295:instrumentation* / Q000379:methods; D009339:Needles; D010316:Particle Size; D010957:Plasmids; D011786:Quality Control; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D014170:Transformation, Genetic; D014783:Viscosity
D004251:DNA Transposable Elements