ythdf3 mutants  (Addgene inc)

Journal: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: (A) Numbers of male and female fish of each genotype. Sibling control and double ythdf2;ythdf3 homozygotes were offspring from the same cross, depicted on top. (B) Gonad histology of double homozygous ( ythdf2 −/− ; ythdf3 −/− ) and sibling control fish from the cross in (A). At 27 dpf, mutants exhibit less developed juvenile ovaries than controls. At 35 dpf, 6 sibling fish had adult ovaries and 8 had testes, while all 12 ythdf2 −/− ; ythdf3 −/− fish had testes. I, stage I oocytes; II, stage II oocytes; triangle, apoptotic oocyte; sg, spermatogonia; sc, spermatocytes. n, replicate number with similar gonads. Scale bars, 40 μm. (C) Numbers of male and female fish of each genotype, following treatment with 17α-ethynylestradiol (EE2). Fish were from the same cross as in (A).

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Control

Journal: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: (A) MZ ythdf2 ;MZ ythdf3 , background-matched wild-type, and unrelated TU-AB wild-type zebrafish embryos develop at similar rates. Parents of mutant and background-matched control embryos were 17α-ethynylestradiol treated. n, replicate number of embryos at same developmental stage. Scale bars, 500 μm. (B) Biplot of expression (log 2 RPKM) of maternal (n = 13,642) and m 6 A-modified (n = 2,280) mRNAs between wild-type and MZ ythdf2 ;MZ ythdf3 , from 6 hpf poly(A) mRNA-seq. Dashed lines, 2-fold change. (C) Cumulative distribution of fold changes in maternal mRNA abundance (log 2 RPKM) between 4 and 0 hpf in MZ ythdf2 ;MZ ythdf3 embryos, for m 6 A-modified (n = 708) and non-modified (n = 841) mRNAs, from poly(A) mRNA-seq. p values computed by a Mann-Whitney U test. (D) Schematic of cross and genotyping strategy for triple Ythdf mutants. Female fish ( ythdf1 +/− ; ythdf2 −/− ; ythdf3 +/− ) were crossed to males ( ythdf1 −/− ; ythdf -+/− ; ythdf3 −/− ) to generate triple homozygotes (1 of 8 possible genotypes). Every 3 days, 48 larvae were genotyped, with 200 more fish genotyped at 30 dpf. (E) Percentage of triple heterozygous (het) or triple homozygous (homo) fish during development. Dotted line, expected percentage (12.5%) of each genotype, from cross in (D). (F) Number of fish with each genotype from cross in (D) at 30 dpf. For each ythdf allele: filled circle, heterozygous; m, homozygous. Dotted line, expected fish number (25), equal for all genotypes.

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Mutagenesis, Control, Expressing, Modification, MANN-WHITNEY

Journal: Cell reports

Article Title: Ythdf m 6 A Readers Function Redundantly during Zebrafish Development

doi: 10.1016/j.celrep.2020.108598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For gene editing to generate ythdf2 −223/−223 and ythdf3 mutants, 30 pg of each sgRNA was co-injected with 150 pg of Cas9 (plasmid pT3TS-nCas9n, Addgene #46757, ( )) capped mRNA synthesized using mMessage mMachine T3 Transcription kit (Thermo Fisher Scientific, AM1340).

Techniques: Recombinant, Nick Translation, SYBR Green Assay, RNA Extraction, Reverse Transcription, Mutagenesis, Software