• PMP22 is predominantly expressed in the compact myelin of the peripheral nervous system, and its levels require strict regulation. Gene duplication-induced mutations of PMP22 lead to CMT1A, which makes up 50 - 70% of all CMT cases.
• Gene editing strategy: The BAC containing the whole human PMP22 genome sequence was randomly inserted into mouse genome in B-Tg(hPMP22) mice.
• mRNA expression analysis: Mouse Pmp22 mRNA were detectable in wild-type C57BL/6 mice and hemizygote B-Tg(hPMP22), Human PMP22 mRNA was detectable only in hemizygote B-Tg(hPMP22) but not in wild-type mice.  
• In vivo efficacy: Human PMP22 targeted nucleic acid drugs (from client) was efficacious in B-Tg(hPMP22) mice.
• Application: This product is used for pharmacodynamics and safety evaluation of CMT1A.

Gene targeting strategy for B-Tg(hPMP22) mice. The BAC containing the whole human PMP22 genome sequence was randomly inserted into mouse genome in B-Tg(hPMP22) mice.

Strain specific analysis of PMP22 mRNA expression in wild-type C57BL/6 mice and B-Tg(hPMP22) mice by RT-PCR. Sciatic nerve RNA were isolated from wild-type C57BL/6 mice (+/+) and hemizygote B-Tg(hPMP22) (Tg/+), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human PMP22 primers. Mouse Pmp22 mRNA were detectable in wild-type C57BL/6 mice and hemizygote B-Tg(hPMP22). Human PMP22 mRNA was detectable only in hemizygote B-Tg(hPMP22) but not in wild-type mice.

The inhibitory efficiency of the nucleic acid drugs against human PMP22 in hemizygote B-Tg(hPMP22) mice. The human PMP22 targeted nucleic acid drugs (provide by client) and saline were administered to the B-Tg(hPMP22) mice individually on day 0. The mice were sacrificed on day 21, and the sciatic nerve tissue was collected to detect the expression level of human PMP22 mRNA by qPCR. The human PMP22 mRNA in the treatment groups (siRNA) were significantly reduced compared to the control groups (saline), demonstrating that B-Tg(hPMP22) mice provide a powerful preclinical model for in vivo evaluation of human PMP22 targeted nucleic acid drugs. Values are expressed as mean ± SEM.

Note: This experiment was performed by the client using B-Tg(hPMP22) mice. All the other materials were provided by the client.

Behavioral performance in wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice. Clasping tests were conducted to assess the behavioral performance in female (A) and male (B) wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice (female, 11-week-old, n=15; male, 13-week-old, n=10). Transgenic B-Tg(hPMP22) mice show significantly higher clinical scores (1~3) versus wild-type mice (~0) , suggesting impaired neurological function. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA. ***P < 0.001.

Behavioral performance in wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice. (A) Grip strength tests were conducted to assess the muscular strength and grip ability of female (A) and male (B) wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice (female, 11-week-old, n=15; male, 13-week-old, n=10). The grip strength of female transgenic B-Tg(hPMP22) mice was decreased compared with wild-type mice, showing grip strength impairments of transgenic female B-Tg(hPMP22) mice. All grip strength measurements are normalized to the individual animal’s body weight. Right, Clinical score of transgenic B-Tg(hPMP22) mice. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA.  *P < 0.05, **P < 0.01, ***P < 0.001.

Behavioral performance in wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice. Rotarod tests were conducted to assess the motor coordination, balance, and endurance of female (A) and male (B) wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice (female, 11-week-old, n=15; male, 13-week-old, n=10). The latency to fall, rodspeed and total distance were decreased in transgenic B-Tg(hPMP22) mice, showing motor impairments of transgenic mice. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA.  *P < 0.05, **P < 0.01, ***P < 0.001.

Gait analysis of wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice. Gait analysis was conducted to assess the locomotor function and coordination of wild-type C57BL/6JNifdc and transgenic B-Tg(hPMP22) mice (male, 13-week-old, n=10). The transgenic B-Tg(hPMP22) mice exhibit significantly different gait characteristics across multiple gait parameters, indicating that the locomotor ability and coordination of the transgenic mice are abnormal. Data were represented as mean ± SEM. Statistical analysis was performed using unpaired two-way ANOVA. * p ≤ 0.05, ** p≤ 0.01, *** p ≤ 0.001.  RF, right front paw. LF, left front paw. RH, right hind paw. LH, left hind paw. 

Comparison of conduction velocity between C57BL/6 and B-Tg(hPMP22) mice. The bar graph illustrates the mean conduction velocity (in meters per second) for wild-type C57BL/6 (male, 8-week-old, n=5) and B-Tg(hPMP22) (male, 8-week-old, n=12). The B-Tg(hPMP22) mice exhibit significantly lower conduction velocities compared to the wild-type C57BL/6, suggesting that the B-Tg(hPMP22) mice have impaired nerve conduction, which is consistent with phenotypes of CMT1A.

Note: This experiment was performed by the client using B-Tg(hPMP22) mice. All the other materials were provided by the client.