The Role Of Human SHOX Gene During Mouse Limb Bud Chondrogenesis

Objective:The mechanism of human short stature homeobox gene (hSHOX) during thechondrogenesis is still not clear. The cartilage formation of mouse limb budmesenchymal cells at E11.5which was infected with lentivirus of hSHOX gene wasevaluated by micromass culture for five days as an in vitro model. The genes related tolimb bud chondrogenesis were detected by real time PCR.Methods:1. Construction of lentiviral vector of hSHOX geneThe cDNA fragment of hSHOX gene from pcDNA3.0was released by restrictionendonuclease and cloned to vector of pBS185, then the whole cassette which includedhCMV promoter and polyA tail was cloned to lentivirus of pNL-EGFP and desinated aspNL-EGFP-hSHOX;2. Confirmation of genes expression of hSHOX and mShox2The DNAs of pNL-EGFP-hSHOX and pNL-EGFP-mShox2were transfected into293T cells. The EGFP expression was verified under fluorescence microscope, thehSHOX and mShox2gene expression were detected by western blot.3. Production of lentiviral praticleThe lentiviral DNAs and packaging plasmids of Helper and VSVG wereco-transfected into293T cells. The supernatant was collected after48hours and viralparticles were recovered by ultra-filtration and concentration. The titer of lentivirus wasdetected.4. Micromass culture of mouse limb bud mesenchymal cells at E11.5The wild type embryo derived from C57/BL mouse and homozygote embryoderived from heterozygote of mShox2knock-out mouse were genotyped by rapid PCR.Limb bud mesenchymal cells at E11.5were separated and infected with lentivirus of hSHOX gene and mShox2gene by micromass culture which a10-μl drop of cellsuspension with the number of105cells was placed in the center of a well. The cultureswere stained with alcian blue after5days for checking the cartilage formation at differentgroups and the left cultures were used for RNA preparation and cDNA transcription.5. Detection of genes related to cartilage formation by Real Time PCRGenes related to cartilage formation such as Runx2,Col X,Col2a,Osteocalin,Ihh andSox9were detected by Real Time PCR which its specific primer was designed accordingto GeneBank and publications.Results:1. Lentiviral vector of pNL-EGFP-hSHOX was successfully constructed andidentified by restriction enzyme digestion, DNA sequencing and western blot.2. hSHOX and mShox2lentiviral particle with high titer were produced.3. The numbers of cartilage formation were significantly decreased by69%(p<0.01)in the group of mShox2knock-out mouse than that of wild type group by micromassculture of limb bud mesenchymal cells at E11.5.4. The numbers of cartilage formation were significantly increased1.5times and1.8times respectively (p<0.01) in the wild type mesenchymal cells at E11.5whichhSHOX and mShox2genes were overexpressed.5. Gene overexpression of hSHOX and mShox2could rescue cartilage formationand the numbers were increased3.5times and3.9times respectively (p<0.01) in limbbud mesenchymal cells with mShox2gene knock-out at E11.5.6. The Runx2and ColX genes were upregulated by12.1and2times duringcartilage formation with overexpression of hSHOX and mShox2genes in mShox2genedeficient limb bud mesenchymal cells at E11.5.Conclusion:1. The numbers of cartilage formation were decreased significantly in mShox2knock-out mouse, overexpression of hSHOX gene could rescue cartilage formation inmShox2gene deficient mouse.2. The Runx2and ColX genes were upregulated during cartilage formation withoverexpression of hSHOX gene and these indicated that hSHOX gene was involved in the differentiation and hypertrophy of mesenchymal cells.3. hSHOX gene shared the similar function with mShox2gene during the limb budcartilage formation.