The Zcwpw1-deficient mouse model in a C57BL/6 genetic background was generated by deleting the genomic DNA fragment covering exon 4 to exon 7 using the CRISPR-Cas9–mediated genome editing system (Cyagen Biosciences, USA). The founders were genotyped by polymerase chain reaction (PCR), followed by DNA sequencing analysis. The mice were housed under controlled environmental conditions with free access to water and food, and illumination was on between 6:00 am to 6:00 pm. All experimental protocols were approved by the Regional Ethics Committee of Shandong University.

Genotyping was performed by PCR amplification of genomic DNA extracted from mouse tails. PCR primers for the Zcwpw1 mutant allele were 5′- AGC TGCTGGGATTAAATGTCTGTTCC-3′ (forward) and 5′-CTACACTGTGCCTTCTACCTTCTTTGAGA-3′ (reverse), yielding a 690–base pair (bp) fragment. PCR primers for the Zcwpw1 wild-type allele were 5′-CAAGATGGAGGAGATATGCAGTACATG-3′ (forward) and 5′-CTACACTGTGCCTTCTACCTTCTTTGAGA-3′ (reverse), yielding a 617-bp fragment.

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