Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

In this video, we present an improved oocyte-specific condition knockout method in zebrafish, offering a versatile platform for studying maternal factors with zygotic mutations resulting in lethality or sterility. Current maternal knockout method are either technically challenging or time-consuming. We had previously reported zpc:cas9 transgene is transcriptionally silenced over generations.

Using zpc:cas9 knock-in line, we have established a robust platform for generating conditional knockout during oogenesis, enabling the rapid and highly-efficient production of maternal mutants. To begin, collect embryos that have been spawned by homozygous rbm24a RFPKI zpc:cas9 fish. Combine one microliter of PGG dust EB rbm24a 4sGRNA with one microliter of Tol2 transposase messenger RNA to prepare the injection mixture.

Dilute both the plasmid and the Tol2 messenger RNA directly in pure water. Now place a glass coverslip in a 90-millimeter dish. Align the embryos along the edge of the coverslip, and gently use a pipette to remove any excess water.

Then inject two nanoliters of the prepared mixture into the blastodisc of each one-cell stage embryo. Gently rinse the injected embryos with blue water, and transfer them to another dish. Place the dish in an incubator set to 28 degrees Celsius for cultivation.

At 24 hours post-fertilization, select embryos that show robust and ubiquitous blue fluorescent protein expression. At four days post-fertilization, raise only the embryos that maintain strong transgenic fluorescent signals. Collect embryos produced from mating mosaic transgenic founder females with wild-type males.

Using a fluorescent stereomicroscope, pick up the blue fluorescent protein-positive embryos at the one-cell stage. Then fix the embryos at appropriate stages in 4%paraformaldehyde at four degrees Celsius overnight. To prepare a 1.5%OGER solution, weigh and combine OGER powder in 1/3-strength Ringer's buffer and boil until fully dissolved.

Pour the melted OGER solution into a 90-millimeter Petri dish and insert a Z-mold into the molten agarose. Once the agarose has solidified, gently remove the mold to create a ready-to-use plate. Next, adjust the settings on the needle puller to use two light weights, and select the step one procedure.

Load the glass capillaries into the puller to make melt-sealed needles. Using pointed tweezers, trim the capillary tips to a 30 to 40-micrometer diameter. Then use a microforge to form a spike at the tip, which will aid in embryo penetration and reduce tissue damage.

Mate the putative female founders with wild-type zebra fish and collect embryos. At the one-cell stage, isolate embryos that are positive for blue fluorescent protein using a fluorescent stereomicroscope. At three hours post-fertilization, incubate the embryos in pronase, dissolved in one third strength Ringer's buffer for 10 minutes with gentle pipetting to dechorionate them.

Carefully transfer the dechorionated embryos onto the prepared argarose plate flooded with 1/3 Ringer's buffer, supplemented with penicillin streptomycin. Reorient the embryos so that the blastomere is facing toward the capillary tip for cell aspiration. Now use a microinjector to aspirate 20 to 40 cells from each embryo by gently reducing the equilibrium pressure to counter the capillary aspiration force.

Increase the equilibrium pressure to release the aspirated cells into two microliters of deionized water at the edge of 1.5-milliliter Eppendorf tubes. Then add 200 microliters of RNA extraction reagent to the tube to wash down the aspirated cells, and place the tube on ice for temporary storage. Keep the embryos after cell aspiration on the agarose plate until aberrant phenotypes are visualized.

Next, add 40 microliters of chloroform to the cells from selected embryos in the extraction reagent and mix gently. Then centrifuge the mixture at 12, 000 G for one minute at room temperature. After collecting the supernatant, add one microliter of glycogen solution.

Then add an equal volume of isopropanol based on the supernatant volume and mix thoroughly. Incubate the tube at minus 20 degrees Celsius for 30 minutes to allow RNA precipitation. Now centrifuge the sample at 12, 000 G for 10 minutes at four degrees Celsius and discard the supernatant.

Then to wash the RNA pellet twice, add 500 microliters of 70%ethanol and centrifuge at 12, 000 G for one minute at four degrees Celsius. After discarding the supernatant, open the tube lid to let the pellet dry at room temperature for five minutes. Add seven microliters of water to dissolve the RNA pellet.

Perform reverse transcription using a First Strand cDNA synthesis kit, then conduct PCR and Sanger sequencing to analyze mutations in maternal mutant embryos. Maternal rbm24a mutants were identified by the absence of rbm24a RFP, while other maternal mutants were detected by specific phenotypes or cell aspiration-based genotyping. Among BFP-positive embryos, those lacking RFP signal were identified as maternal rbm24a mutants.

In situ hybridization using NANOS3 probe revealed that Mrbm24a embryos failed to recruit germ plasm mRNAs to germ granules at the four-cell stage, and lacked primordial germ cells at 24 hours post-fertilization. All adult Mrbm24a males failed to fertilize eggs spawned by wild-type females, showing anatomical abnormalities with fatty deposits replacing normal testes. Histological analysis confirmed the complete absence of germ cells and spermatozoa in Mrbm24a testes.

Western blot analysis confirmed nearly undetectable levels of rbm24a protein in BFP-positive RFP-negative embryos. RT-qPCR results showed significantly lower rbm24a transcript levels in RFP-negative BFP-positive embryos relative to controls. RT-PCR and Sanger sequencing revealed large deletions and indels in both RFP-negative BFP-positive and RFP-positive BFP-positive embryos with wild-type transcripts detectable only in RFP-positive embryos.

A maternal GFP marker and nanog sgRNAs were introduced to generate maternal nanog mutants, and GFP-positive embryos showed a range of dorsalized phenotypes. Germline transmission rates ranged from 12%to 32%among GFP-positive transgenic lines. A dorsal phenotype consistent with maternal nanog mutants was observed in 22%to 60%of GFP-positive embryos.