• The transmembrane serine/threonine kinase receptor known as ACVR2B (activin A receptor type IIB) is an essential part of the transforming growth factor-β (TGF-β) superfamily signaling pathway. It binds ligands like growth differentiation factor 11 (GDF11), myostatin, and activins to control a variety of physiological processes. It has cell-type-specific expression and is involved in erythropoiesis, adipogenesis, folliculogenesis, and muscle growth and maintenance.
• Gene editing strategy: The exons 1-10 of the mouse Acvr2b gene that encode the whole ，molecule, including promoter, 5’UTR, and part 3’UTR, were replaced by the exons 1-11 of the human counterparts, including human promoter, 5’UTR, and human 3’UTR in B-hACVR2B mice. The human ACVR2B expression is driven by the human ACVR2B promoter, while the mouse Acvr2b gene transcription and translation will be disrupted.
• mRNA Expression Analysis: Human ACVR2B mRNA was exclusively detectable in B-hACVR2B mice but not in wild-type mice. Mouse Acvr2b mRNA was exclusively detectable in wild-type mice but not in B-hACVR2B mice.
• Protein Expression Analysis: ACVR2B protein was detectable in the gastrocnemius, brown fat, and inguinal fat of homozygous B-hACVR2B mice.

Gene editing strategy for B-hACVR2B mice. The exons 1-10 of the mouse Acvr2b gene that encode the whole molecule, including promoter, 5’UTR, and part 3’UTR, were replaced by the exons 1-11 of the human counterparts, including human promoter, 5’UTR, and human 3’UTR in B-hACVR2B mice. The human ACVR2B expression is driven by the human ACVR2B promoter, while the mouse Acvr2b gene transcription and translation will be disrupted.

Strain specific analysis of ACVR2B mRNA expression in wild-type C57BL/6JNifdc mice and B-hACVR2B mice by RT-PCR. Gastrocnemius RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hACVR2B mice (H/H) (male, 8-week-old), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human ACVR2B primers. Human ACVR2B mRNA was exclusively detectable in B-hACVR2B mice but not in wild-type mice. Mouse Acvr2b mRNA was exclusively detectable in wild-type mice but not in B-hACVR2B mice. Human sequences were confirmed via Sanger Sequencing.

Species specific analysis of mouse and human ACVR2B gene expression in wild-type C57BL/6JNifdc mice and homozygous B-hACVR2B mice by RT-qPCR. Gastrocnemius RNA was collected from wild-type C57BL/6JNifdc mice (+/+) (male, n=1, 8-week-old) and homozygous B-hACVR2B mice (H/H) (male, n=1, 8-week-old), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human ACVR2B primers. Human ACVR2B mRNA was exclusively detectable in B-hACVR2B mice but not in wild-type mice. Mouse Acvr2b mRNA was exclusively detectable in wild-type mice but not in B-hACVR2B mice. Values are expressed as mean ± SEM.

Western blot analysis of ACVR2B protein expression in homozygous B-hACVR2B mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hACVR2B mice (H/H) (male, 8-week-old), and then analyzed by western blot with anti-ACVR2B antibody (Abcam, ab76940). 40 μg total protein was loaded for western blotting analysis. ACVR2B protein was detectable in the gastrocnemius, brown fat, and inguinal fat of homozygous B-hACVR2B mice and wild-type C57BL/6JNifdc mice, as the antibody cross-recognizes both human and mouse ACVR2B.

The inhibitory efficiency of the nucleic acid drugs against human ACVR2B in homozygous B-hACVR2B mice. B-hACVR2B mice (H/H) were randomly divided into two groups (male, 8-week-old, n=3). The human ACVR2B targeted nucleic acid drugs and the PBS were administered to the mice individually. The mice were sacrificed on day 14, and the inguinal subcutaneous fat and quadriceps femoris tissue were collected to detect the expression level of human ACVR2B mRNA by qPCR. (A) The schematic diagram of experimental processing. (B) The expression of human ACVR2B mRNA was normalized to the PBS group of each tissue. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001.

Note: This siRNA from a client target ACVR2B in adipose and muscle. Data are shared upon the client's approval.