Generation Of DSPP Transgenic Mouse Models And Related Studies

Dentin sialophosphoprotein(DSPP), which was considered to be cleaved into dentin sialoprotein(DSP) and dentin phosphoprotein (DPP) at posttranscriptional level, belongs to non-collagenous protein(NCP), and got its name by the fact that it was first discovered to be secreted by odontoblasts and appear at the mineralization front of dentine. It was once regarded as the unique dentine-specific protein, but according to recent studies, it was also expressed in other tissues and organs, such as bone, cartilage, and even inner ear, which means that perhaps it was not a dentine-specific protein, and it might have other functions rather than involving in the mineralization process of tooth. To further the studies about the role of DSPP in the development and mineralization of tooth, and start the researches about the functions of DSPP in other tissues and organs, attempts of the generation of in vivo biomodel organism for the function analysis of DSPP were carried out. The most important task of the plan is to establish DSPP transgenic and knockout mice models. The followings were parts of this plan, including the generation of DSP transgenic mice, DSPP tetracycline-responsive transgenic mice system, the construction of DSPP knockout construct and the attempt on DSPP knockout by ES cell gene targeting.1. Generation of DSP transgenic micepcDNA3.1 -CX was constructed by substituting CMV promoter of pcDNA3.l with c β -actin promoter, and the ultimate transgenic construct was established by cloning DSP coding sequence(with a HA-Tag linked to its C-terminal) into pcDNA3.1-CX. The transgenic DNA was linearized by BgIII digestion and then microinjected into the male pronucleus of the mouse zycotes. The tail DNA of pups was tested by PCR and Southern blot. 4 founders were characterized and breeding process was carried out. RT-PCR showed that the transgene was expressed in many organs.2. Generation of tetracycline-responsive transgenic mice system of DSPPThe promoter of pTet-on-NSE was replaced by c β -actin promoter, which led to the construction of the tetracycline- responsive vector pTet-on-CX; and the DSPP transgenic vector pTRE2-DSPP, was constructed by subcloning DSPP coding sequence from pBlueScript-DSPP to pTRE2. Candidates of the transgenic mice were generated by microinjection and pups were screened by PCR and Southern blot. All together 11 and 10 founders were got, respectively; and breeding process was carried out. RT-PCR showed that the transgene pTet-on-CX could be expressed in many organs.3.Cloning of DSPP specific promoter and generation of DSPP-specific -promoter-LacZ transgenic mice.According to the sequence reported by MacDougall et al, primers for DSPP specific promoter were designed, and PCR was performed. A 1.6 kb fragment was obtained and it was confirmed to be DSPP specificpromoter by sequencing. DSPP-specific-promoter-LacZ transgenic construct was constructed by subcloning, and transgenic mice were produced bymicroinjection and screened by PCR. 12 founders were obtained.4. Attempts on DSPP knockout by ES cell gene targetingThe primers were designed by the use of DNAstar software and were in accordance with the sequence provided by NCBI GenBank. Using genomic DNA of ES cells as template, PCR was performed to get the upper and lower homology arms of the targeting constructs. After characteration by sequencing, the homology arms were subcloned, together with the screening marker, e.g. hsv-tk-neo and PGK-TK, into pBluescript in a definite order. The ultimate constructs were confirmed by enzyme digestion and sequencing, and were linearized by NotI for the use of electrotransformation of the ES cells. Altogether 2 targeting constructs were constructed, 3 transformation experiments were performed and 374 ES cell clones were tested by PCR and Southern blot, no positive clone was found up to date.The generation of DSP and DSPP transgenic mice, and attempts on DSPP gene knockout make basis for further function analysis of DSPP.