The Establishment Of HSP22 Gene Transgenic Mouse Model

Charcot-Marie-Tooth disease(CMT),also known as hereditary motor and sensory neuropathy(HMSN),is the most common hereditary peripheral neuropathy with highly clinical and genetic heterogeneity. CMT is characterized by progressive and symmetric distal muscle atrophy,weakness,hyporeflexia and hypoesthesia in distal limbs.The incidence of CMT is about 1/2500.According to the electrophysiological and histopathological criteria,CMT can be divided into two forms:the demyelinating form(CMT1)and the axonal form(CMT2).Several modes of inheritance have been described in CMT,including autosomal dominant(AD),autosomal recessive(AR)and X-linked dominant or recessive(XD/XR).With the advancement of the biogenetics,so far 32 gene loci have been identified and 22 distinct genes have been cloned.In 2004,we found a large Chinese CMT2 family in Hunan and Hubei provinces which was proved to be a novel genotype designated as CMT2L.After mapping the locus to a 6.8cM region at chromosome 12q24.2-12q24.3,we identified a novel 423G→T mutation of HSP22 gene and confirmed amino-acid Change K141N as the causative gene defect in CMT2L.Although many mutations of a number of genes have been associated with CMT,the disease mechanisms caused by each gene may be different.The main mechanisms include dosage effect,loss of function, gain of function or dominant-negative effect.Many researches have found that HSP22 not only can interact with HSP27,but may be involved in the modulation of HSP27 activities.The K141N mutation in the HSP22 protein does not disrupt interaction with HSP27,but strengthen it, leading to the formation of aggregates.So,the K141N mutation may have a gain-of-function and cause CMT.To confirm the presumption,we can construct the transgenic mice which carry the K141N mutation of human HSP22 gene through microinjection.Recently studies showed that HSP27 gene mutations could also be associated with CMT.The phenotypes caused by HSP22 or HSP27 are similar.This would be a good clue not only for the researches about the normal function of sHSPs,but the researches about the pathogenesis of CMT.By over expression of heat shock protein 27(HSP27)in transgenic mice,many scholars have found that HSP27 not only could reduce cell death in the hippocampus associated with seizures and reduce infarct size in cardiac or brain ischemia but has significant cytoprotective properties in rescuing nerve injury,maintaining muscle function.Whether HSP22 has the similar properties as HSP27,there are still no relevant reports and no transgenic mice over expression HSP22.It is necessary to establish the transgenic mice model over expressing HSP22 for improving our understanding the normal function of HSP22 and learn more about the pathogenesis of HSP22 mutations.To construct the transgenic mouse models carrying the mutation K141N of human HSP22 gene or the wild-type human HSP22 gene,we constructed pCAGGS-HA-^K141NHSP22 and pCAGGS-HA-^wtHSP22 transgenic expression vectors,pCAGGS-HA-^K141NHSP22 and pCAGGS-HA-^wtHSP22 could express effectively in eukaryocytes such as COS7 and HEK293T cells through confocal microscopy and western blot analysis.We also confirmed that mutant HSP22 protein formed large aggregates predominantly located around the nucleus and wild HSP22 protein distributed diffusively in the cytosol and nucleus.A prospective 25.5kDa protein band of HA-tagged HSP22 could be specificity detected by Western blot with a monoclonal antibody to the HA tag.Our results showed that pCAGGS-HA-^K141NHSP22 and pCAGGS-HA-^wtHSP22 could be used to further transgenic researches.The linearized DNA was cut out of the pCAGGS-HA-^K141NHSP22 or pCAGGS-HA-^wtHSP22 vectors by SalI,HindⅢand BsaⅪI digestion,purified and used to generate the transgenic mice.The linearized pCAGGS-HA-^K141NHSP22 was microinjected into 640 fertilized eggs from C57BL mice,which were subsequently transferred to pseudopregnant recipients.24 babies were produced.Among them,4 transgenic founder mice(Tg642,Tg677, Tg679,Tg681)carrying the mutation K141N of human HSP22 gene were identified by PCR and direct sequence.Tg642 can breed very well and have produced 23 F1 offsping.Among them,4 transgenic F1 mice (Be423,Be435,Bf84,Bf86)carrying the mutation K141N of human HSP22 gene were identified by PCR.But the Tg642 line mice didn't express the human HSP22 protein and can not be used for further researches.The linearized pCAGGS-HA-^wtHSP22 was microinjected into 210 fertilized eggs from C57BL mice.19 babies were produced.Among them,4 transgenic founder mice(Tg646,Tg648,Tg649,Tg661)carrying human HSP22 gene were identified by PCR and direct sequence.We will analyze the expression of exogenous mutant HSP22 protein if we can get more offspring carrying the mutation K141N of human HSP22 gene.If our transgenic mice express the human mutant HSP22 protein,we will observe whether they have the similar behavior, electrophysiology or pathology features as human CMT2L disease.This will establish foundations for the pathogenesis and therapy researches of CMT.Similarly,if the transgenic founder mice carrying the human HSP22 gene could breed well,we will analyze whether these mice could over express human HSP22 protein.We could analyze the neuron protective function of HSP22 in the transgenic mice over expression of HSP22 protein.Many sHSPs are often upregulated and accumulate into inclusion bodies in many neurodegenerative diseases.This suggests that sHSPs may be potent suppressors of neurodegeneration.Some transgenic mice of neurodegenerative disease such as Alzhermer's disease(AD), Parkinson's disease(PD)or Amyotrophic Lateral Sclerosis(ALS)have been generated.We could make those transgenic mice also to over express HSP22 by mating them with our HSP22 over expressed transgenic mice.Then we can observe whether HSP22 have an effect on the pathological changes on the transgenic mice of neurodegenerative disease.This will provide a new idea about the therapy for those neurodegenerative diseases.