Establishment of a mouse model of spinal muscular atrophy and its biological characteristics
Objective To construct a mouse model of spinal muscular atrophy(SMA)by mating breeding and genotype identification of heterozygous genetically engineered mice,and to analyze the biological characteristics of the constructed off spring SMA mice.Methods SMA model mice were obtained by mating and breeding of FVB.SMNΔ7 hybrid mice(8 females,4 males).The amplified product of motor neuron survival protein 1(SMN1)gene was obtained by polymerase chain reaction(PCR)through the mouse tail genome DNA template of the offspring mice,and the genotype was identified by agarose gel electrophoresis to screen out the SMA model mice.According to the results of genotype identification,the mice were divided into three groups:wild type,heterozygote and SMA.The body and muscle development of neonatal SMA mice were measured by righting reflex test and muscle fiber density measurement(n=5).The ontogeny of offspring mice was evaluated by body mass measurement,righting reflex test and survival curve analysis at different developmental time points(n=6).The expression of SMN protein in spinal cord was detected by immunohistochemistry(IHC),immunofluorescence(IF)and Western blot(WB)(n=3).Results Genetic tests showed that about 25％(8/32)of the offspring of FVB.SMNΔ7 heterozygous mice were homozygous SMN1 mutant mice,namely SMA mice.The righting reflex.time of SMA mice was longer than that of heterozygous mice and wild-type mice(t values:7.48 and 16.48,respectively,both P＜0.05).The muscle fiber density of the hind limbs of mice showed that the number of muscle fibers per unit cross-sectional area of SMA mice was lower than that of wild-type mice and heterozygous mice(both P＜0.05).The body mass of SMA mice at day 0,1,7,10 and 13 was lower than that of wild type and heterozygote mice(all P＜0.05).And at different time points,the ring-over reflex time of offspring SMA mice was longer than that of wild-type and heterozygous mice(all P＜0.05).The median survival time of SMA mice was 14.80±2.60 d,which was lower than that of wild-type and heterozygous mice(both P＜0.05).The IHC results showed that the expression level of SMN protein in the spinal cord of SMA mice was lower than that of wild-type and heterozygous mice(t values:41.78 and 15.36,respectively,both P＜0.05).IF and WB results also showed that the expression level of SMN protein in the spinal cord of SMA mice was lower than that of wild-type and heterozygous mice(both P＜0.05).Conclusions In this study,a mouse model of SMA has been successfully constructed.Offspring SMA mice have shown poor physical and muscle development,ontodevelopmental delay,and decreased SMN protein expression in the spinal cord,which are consistent with the pathological manifestations of SMA.
Muscular atrophy,spinalModels,animalMice,transgenicSurvival of motor neuron 1 proteinSurvival of motor neuron 2 protein
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