Fig. 3

(A) Strategy combining phenotyping and genotyping. Cells are isolated from GFP-positive embryos at 3 hpf and stored in TRIzol. When donors developed maternal mutant phenotype, total RNAs from isolated cells are extracted and analyzed by RT-PCR. Primers are designed to amplify the entire wild-type CDS. PCR products are either analyzed by gel electrophoresis or subcloned into pCS2-MT vector, generating a mini expression library. A control library is made from a wild-type embryo using the same set of primers. These primers ensure the in-frame ligation of the wild-type CDS and the myc epitopes. The libraries are either injected to express protein or subjected to sequencing. (B) RT-PCR analysis of nanog CDS region from 16 Mnanog embryos shows the presence of truncated transcripts. Red asterisks and numbers indicate PCR products subjected to Sanger sequencing. F2 and R2 represent primers to amplify the CDS region. The positions of sgRNA targeting sites are marked as sg1 to sg3. Gray boxes indicate UTRs, and the green represents the CDS region. (C) Diagram summarizes the sequencing results. Dashed lines and red boxes represent deletions and insertions, respectively. (D) Analysis of genomic deletions in Mnanog mutants using different primer sets as displayed on the nanog gene. Grey and black boxes designate UTR and CDS regions of exons, respectively; thin lines indicate introns. (E) Sequencing of PCR products reveals various patterns of genomic deletions. (F) Absence of Nanog protein in verified Mnanog embryos at 3 hpf. (G) Injection of expression libraries constructed from two Mnanog mutants produced no myc-tagged protein product. (H to K) ISH was used to examine the absence of nanog transcripts in Mnanog mutants among GFP-positive embryos at the sphere stage. Scale bar, 250 μm.