Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.1 Microinjection Methods for Anopheles EmbryosPage 1 of 8Chapter 3: Specific Anopheles Techniques3.1 Embryonic Techniques3.1.1 Microinjection Methods for Anopheles EmbryosMark BenedictIntroductionWe present two methods that have been successful (and a variation of Method 1 using oil). The first wasdeveloped by John R. (Randy) Clayton for injection of An. gambiae embryos, and it has been used toobtain high frequency egg hatching and EGFP transient expression rates as described by Grossman etal., 2001 (Grossman et al. 2001). This method is similar to that used by Dave O’brochta’s group at theUniv. Maryland. They successfully transformed An. gambiae using a similar method by covering theembryos with halocarbon oil prior to injection (Kim et al. 2004). Using oil should provide better visibility ofthe needle flow rate.The second method is fast and requires less judgment than those above. It was developed by HervéBossin and Mark Benedict for An. arabiensis and An. gambiae. However, it should be useful for manymosquito species. Anecdotally, mosquito species vary in the ease with which they can be microinjected.Both of the methods above have been used successfully with gambiae s.l. which is supposed to be one ofthe more difficult to inject.We recommend mounting the injection needle in a fixed position and moving the slide holding the alignedembryos using the stage controls to the appropriate place for injection. This allows one to use a rathersimple needle positioner mounted on or by the microscope.Anopheles embryos cannot be dechorionated, so use of rigid needles and firm positioning of the embryosis necessary. Quartz glass injection needles are by far preferable to aluminosilicate or borosilicateneedles. These require higher pulling temperatures than the other glasses and therefore a laser needlepullermust be used.Materials:• Mated adult females bloodfed 3-5 days post-eclosion.• Clean water in a wash bottle• Pipettor e.g. P20• Fine paint brushes 1 and forceps• Filter paper• 2X Na phosphate Injection buffer (see below for preparation; requires KCl and di- and monobasicsodium phosphate)• Minimum fiber filter paper e.g. (Whatman 1450-090, ‘Hardened circles’)• Eppendorf Microloader tips (no. 5242-956-003)• Ultrafree-MC filters (no. UFC30HV00)• Quartz glass capillaries, 1 mm OD, 0.7 mm ID X 10 cm length (e.g. Sutter no. QF100-70-10 )1 Select the brushes carefully from among the finest at an art or craft store. Sable brushes are excellentand more expensive, but regardless, a very fine pointed tip is essential.