Dp214

ChIP assays were performed on HTR8/SVneo cells stably overexpressing KLF9 using a ChIP assay kit (P2078, Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions. HTR8/SVneo cells (2×10⁶ cells) were cross-linked in 10 cm dishes, then the nuclear lysates were sonicated to shear DNA to approximately 200–300 bp fragments, followed by immunoprecipitation overnight at 4 °C using the anti-IgG or anti-KLF9 antibodies listed in Supplementary file 4. The immunoprecipitated DNA was purified using a DNA purification kit (DP214, Tiangen Biotech Co.,Ltd, China) and then analyzed by PCR using the primers specified in Supplementary file 3.

To evaluate the gut microbiota, feces were collected from all mice at the 1^st, 3^rd, 6^th, 12^th, and 18^th week and T-RFLP analysis performed. Bacterial DNA was amplified with bacterial 16S rRNA gene-specific primers: 8F (5’-FAM-AGAGTTTGATCATGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) [58 (link)], which included a FAM label at the 5’end of the 8F primer. The PCR products were purified with an agarose gel recovery kit (DP214, Tiangen, China) according to the manufacturer's instructions. Restriction digests were performed with MspI (Hpa, Hap II) (Takara, Dalian, China) according to the manufacturer's instructions. PCR products (20 μL) were incubated for 4 hr at 37°C followed by 80°C for 20 min. The fragments (T-RFs) were desalinated by ethanol precipitation and then mixed with an internal size standard (LIZ500) at 95°C for 5 min. The fragments were sequenced with a DNA Sequencer in the range of 50–1000 bp (ABI PRISM 3700, USA) and the results analyzed using the Peak Scanner (v1.0) and Gene Marker 2.20 software.

The production process for the ASMT-overexpressed goats is shown in Figure 1A. Healthy Laoshan dairy goat ewes with a body weight about 65 kg were selected and primed with progesterone using a controlled internal drug release (CIDR) device (EAZI-BREED^® CIDR^® Sheep and Goat Device, Auckland, New Zealand) for estrus synchronization. Excellent robust rams were used for artificial semen collection. Superovulation and endoscopic-assisted insemination were carried out on embryo-donor goats. The plasmids were digested with NotI and SalI enzymes, and the linearized DNA was extracted from the gel and was purified with a DNA purification kit (Tiangen, Beijing, China, DP214). The linearized DNA solution was then injected into the cytoplasm of the prokaryotic embryos at a concentration of 10 µg/mL and a volume of 5 pL. A total of 3 to 5 embryos that were in good condition were transferred to one recipient within one hour. A total of 55 recipients were transplanted. After 60 days, a B-ultrasound was performed to examine the pregnancy status of the recipient. A total of 18 recipients became pregnant, and 10 lambed.