| BackgroundCleft palate is one of the most common of human birth defects in infancy , which cause disrupting facial structures, with an incidence of about 1/1 000 newborns worldwids every year. Cleft palate arise from many etiologies, including single-gene disorders, multiple genetic factors disturbance, chromosome aberrations, exposure to teratogens, and sporadic conditions of unknown cause. The causes resulting in cleft palate are being dissected through studies of human populations and through the use animal models. Mouse models in particular have made a substantial contribution to our understanding of the gene pathways involved in palate development and the nature of signaling molecules that act in a tissue-specific manner at critical stages of embryogenesis.Very recently studies have shown that Titf-2 gene play a important role in palatine shelft embryonic development in human and in mouse. Titf-2 gene, which encode thyroid transcription factor-2( TTF-2), is located on chromosome 9q22 in human and located on chromosome 4C2 in mouse. Titf-2 gene consists of a single exon encoding for a 42kDa protein that is phosorylated and contain an alanine stretch of variable length. The mutation of Titf-2-/- in human and in mouse can result in cleft palate in infancy. However, whether the mutation of Titf-2+/+ consistently expression may induce the cleft palate in human and in mouse is still unknown. So, our objective is to establish the Titf-2 transgenic mouse model with cleft palate by gene microinjection and to investigate the possible relationship between the mutation of Titf-2 gene and the cleft palate in embryonic development in mouse.Methods1. To induce the over ovulation with 6 week-old-female mouse by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (HCG).2. To prepare the phantom pregnancy mouse through copulating with ligatedmale mose.3. To harvest fertilized ovums at different time.4. To amplificate the recombinated mouse Titf-2 gene, which was obtainedfrom Dr. Huang, by Escherichia coli.5. To inject the titf-2 gene into the fertilized ovums by microinjection.6. To culture the fertilized ovums with Titf-2 gene up to developing into atwo-cell embroy.7. To transplate the two-cell embroy into a fallopian tube of phantom pregnancy mouse.8. To observe the palate shelft embroynic development in normal mouse andTitf-2 transgenic mouse.9. To analyse the integrating of the objective gene into mouse genome.Results1. Quantity and quality of male-pronucleus of fertilized ovums harvested at25～27h after injecting HCG was much higher than that at other time.2. The 982 fertilized ovums were injected with Titf-2 gene,in which there wereabout 580 fertilized ovums developing into two-cell embroy.3. There were 20 phantom female mice to be transplanted the two-cell embroy,in which there were 16 mice to have pregnacy,and the gestated rate was about 80%.4. The palate shelft of normal fetal mouse began to coalesce at E15. But thepalate shelft of Titf-2 trangenic mouse did not.5. 3 F1 mice and 41 F1 fetal mice were harvested after transplatation of two-cell. There were 3 F1 mice with cleft palate and 2 with fetal mice with cleft palate.6. 3 F1 with cleft palate were positive Titf-2 transgenic expression and 3 F1fetal mice were positive Titf-2 transgenic expression by PCR analysis.7. 3 F1 mice with cleft palate and 3 F1 with cleft palate were positive expression by Southern Blot. So ,false positive rate of PCR analysis was about 60%.Conclusions 1. The optimal time of male-pronucleus of fertilized ovums for microinjection is at 25～27h after injection of HCG.2. The palatine process of the normal mice began to coalesce at E15 and the cleft palate of Ttif-2 transgenic mouse did not.3. The false positive rate of Titf-2 expression can be decreased by analysis method combinated PCR and Southern Blot.4. The consistangty expreses of Titf-2 gene may a important roel in cleft palate formation in fetal mouse.5. Titf-2 transgenic mouse model with cleft palate were established by injecting Titf-2 gene into fertilized ovums.
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