Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

This protocol is excerpted from Catrina et al., Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers, J. Vis. Exp. (2019).

1. Design of MBs for Live Cell Imaging

1. Fold the target RNA sequence to predict the mRNA target's secondary structure using the "RNA form" from the mfold server (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form).
   1. Paste/upload the target sequence in FASTA format, select 5 or 10% sub-optimality (structures with a free energy of folding within 5 or 10% of the MFE value, respectively), and adjust the maximum number of computed foldings accordingly (e.g. larger for 10% sub-optimality).
      NOTE: Inclusion of sub-optimal secondary structures when designing MBs allows for the identification of regions within the target mRNA that may be more flexible or more rigid than as predicted for the minimum free energy (MFE) structure alone, which improves the overall design of MBs suited for live cell imaging.
   2. Select an "immediate job" for mRNA targets of 800 nucleotides (nt), or a "batch job" for mRNA lengths between 801 and 8,000 nt. Save the "ss-count" file as simple text file.
2. Use the "ss-count" file obtained in step 1.1 as input for the PinMol program (https://bratulab.wordpress.com/software/) with the desired parameters, to design several MBs for the mRNA target (see tutorials describing usage of PinMol program at https://bratulab.wordpress.com/tutorial-pinmol-mac/).
   1. Determine the specificity of selected MBs by performing BLAST analysis: use "blastn" with the appropriate database (e.g. for oskar mRNA-specific MBs use the "refseq-rna" database and the Drosophila melanogaster organism).
   2. Identify any tissue-specific expression of mRNA target (e.g. for oskar mRNA Flybase> High-Throughput Expression Data> FlyAtlas Anatomy Microarray or modENCODE Anatomy RNA-Seq; http://flybase.org/reports/FBgn0003015) and compare with any positive BLAST hits. Eliminate probes that show >50% cross-homology with other mRNAs that are also expressed in the tissue/cell of interest.
3. Select the fluorophore and quencher pair appropriate for the microscopy set-up available to perform live cell imaging (e.g. Cy5/BHQ2).

2. MB Synthesis, Purification, and Characterization

1. Use in-house synthesis and purification as previously described in Bratu, Methods in Molecular Biology (2006), or services from commercial providers, to synthesize and purify one to five MBs (see above note), using the following labeling scheme: [5'(Fluorophore)-(C3 or C6 linker)-(2'-O-methyl MB sequence)-(Quencher)3']. Purify MBs using reverse-phase HPLC, in house or using the services of the commercial provider.
   NOTE: The phosphoramidites used for automated probe synthesis must have the 2'-O-methyl ribonucleotide modification. One can also use chimeras of alternating locked-nucleic acid (LNA) and 2'-O-methyl modifications to increase the stability of a hybrid between a shorter MB and its target mRNA.
2. Synthesize DNA oligonucleotides that match the sequence of the targeted RNA region and thus are complementary to the probe region of MBs, for use in in vitro characterization (see steps 2.3 to 2.5; above note). Maximize hybridization of the MB with the DNA-oligonucleotide target mimic, by including on each end of the DNA target four additional nucleotides, as found in the target mRNA sequence.
   NOTE: A more rigorous characterization of the MB's efficiency to detect the targeted sequence can be performed using in vitro synthesized RNA targets instead of complementary DNA oligonucleotides.
3. Perform thermal denaturation of the MB alone, measure its melting temperature (Tm), and confirm that the MB assumes the desired hairpin shape at physiological temperature. We have observed Tm values between 60 and 90 °C.
4. Perform thermal denaturation of the MB in the presence of the DNA oligonucleotide target and measure the MB:DNA target hybrid's Tm, as previously described in Bratu, Methods in Molecular Biology (2006). A Tm between 55 and 60 °C is desired for the MB:DNA hybrid.
5. Perform in vitro hybridization reactions with the corresponding DNA oligonucleotide target, and determine the efficiency of MB:DNA hybrid formation at physiological temperature, as previously described in Bratu, Methods in Molecular Biology (2006). Fast hybridization kinetics with the DNA target mimic is desired, however MBs that do not show high hybridization efficiency with DNA targets may have a better performance with the target mRNA in vitro and/or in vivo.

3. Dissection and Preparation of Individual Egg Chambers for Microinjection

1. Feed newly hatched, mated females for 2-3 days with fresh yeast paste.
2. Anesthetize flies on a CO₂ pad and, using fine tweezers (Dumont #5), transfer 1-2 females into a drop of Halocarbon oil 700 on a glass cover slip.
3. Using a pair of tweezers, orient the fly with the dorsal side up under a stereomicroscope. Dissect the female abdomen by making a small incision at the posterior end and gently squeeze the pair of ovaries into the oil.
4. Explant the ovaries onto an oil drop on a new coverslip. Gently hold one ovary with one tweezer while pinching off the youngest stages of the ovariole with the other tweezer. oskar mRNA is actively localized at and after mid-oogenesis (stages > 7), and younger egg chambers (stages < 7) are more difficult to inject and do not survive as long. Slowly drag on the cover slip (with a downward movement) until individual ovarioles or egg chambers are isolated and aligned vertically. Further separate single egg chambers by displacing the unwanted stages from the ovariole egg chain.
   NOTE: Ensure that individually teased egg chambers do not float in the oil, and that they adhere to the cover slip. This is important for both successful microinjection and image acquisition.

4. Microinjection of MBs into the Nurse Cells of Egg Chambers

1. Prepare the MB solution, using one molecular beacon (e.g. osk2216Cy5), or a mix of two MBs that target different mRNAs and which are labeled with spectrally distinct fluorophores (e.g. osk2216Cy5 and drongo1111Cy3). Use a concentration of 200-300 ng/µL each MB in HybBuffer (50 mM Tris-HCl – pH 7.5, 1.5 mM MgCl₂ and 100 mM NaCl). For a cocktail of four MBs labeled with the same fluorophore that are targeting the same mRNA at 200 ng/µL each in HybBuffer (e.g. osk82, osk1236, osk2216). Spin down the MB solution immediately prior to loading the needle for microinjection.
2. Select the objective. A 40x oil objective is recommended for finding an appropriate egg chamber and for performing microinjection.
3. Mount the coverslip with dissected egg chamber onto the microscope stage. Bring up the objective in the focus position and identify an egg chamber at a mid-to-late developmental stage, that is properly oriented for microinjection (i.e., with the AàP axis perpendicular to the needle tip to allow for easy injection within a nurse cell proximal to the oocyte).
4. Load a needle (commercial or prepared in house) with ~1 µL MB solution (see step 1.1) and connect it to the microinjector. For microinjections in D. melanogaster egg chambers, orient the needle (see Table of Materials) at an angle <45° to the microscope stage (e.g. 30°) to avoid puncturing several nurse cells.
5. Set-up the injector with injection pressure of 500-1,000 hPa and compensation pressure of 100-250 hPa (see Table of Materials).
6. Slowly move the stage to bring in the field of view an area of the oil drop void of egg chambers.
7. Using the micromanipulator joystick, gently lower the needle into the oil drop and bring its tip into focus towards the periphery of the field of view.
8. Perform a 'clean' function to remove the air from the tip of needle and to ensure that there is flow from the needle.
9. Bring the needle to the home position and focus on the egg chamber to be microinjected, then bring the needle back into focus and position it near the edge of the egg chamber.
10. Perform a fine adjustment of the objective's Z-position such that the membrane separating the follicle cells from nurse cells is in focus.
11. Insert the needle into a nurse cell and perform injection for 2-5 s.
12. Gently remove the needle and retract it to the home position.
13. Change the objective to the desired magnification for image acquisition (60-63x or 100x), focus on the egg chamber, and begin acquisition.