Target clone plus

The primers used in the experiment are shown in Supplementary Materials Table S2. First-strand cDNA was synthesized from the total RNA isolated from the stem with ReverTra Ace (TOYOBO Co. Ltd., Osaka, Japan) using an adaptor primer for 3′-RACE at 42 °C for 1 h. The cDNA product was then subjected to PCR amplification with KOD-plus-NEO (TOYOBO Co. Ltd.) using a primer set of PEPCF and PEPCR. The PCR amplified fragment (1800 bp) was cloned into pTA2 vector using TArget Clone^™-Plus-(TOYOBO Co. Ltd.) and sequenced. The sequence of a fragment was determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA), and the fragment was sequenced in an ABI 3130xl Genetic Analyzer (Applied Biosystems). For subsequent RACE experiments, primers were designed based on the fragments’ sequences obtained previous step, using a GeneRacer kit (Invitrogen, Waltham, MA, USA). The primers used for the 5′-RACE were insert used for reveres transcription, for the first round, and for the nested PCR. The primers used for the 3′-RACE were adaptor primers for reverse transcription, for the first round, and for the nested PCR. In both cases, the final products were cloned into pTA2 vector using TArget Clone^™-Plus-(TOYOBO Co. Ltd.) and sequenced.

Tail lysates of pups were prepared by an alkaline lysis method and PCR was performed using KOD FX with each primer sets. For the analysis of Il11 mutants, the IL11 F and R primers listed in Table S3 were used according to a previous report (Nakagawa et al., 2015 (link)). Each PCR product was analyzed by direct sequencing. For the analysis of Spp1 gene, PCR were carried out with 37 cycles, and then the products were analyzed as described in the same way as the single blastocyst assay. For the analysis of Gpcpd1 gene, three kinds of PCR were carried out with 37 cycles, and then the products were analyzed as described in the same way as the single blastocyst assay. The PCR1 products harboring floxed mutations were subcloned into a pTA2 vector using Target Clone -Plus- (Toyobo). Each subcloned vector was analyzed by sequencing.