Foundation Of Transgenic Mice Expressing Doppel Without Glycosyl Phosphatidyl Inositol

Exogenous gene is injected into fertilized ovum by micromanipulation instrument, which is integrated into the chromosome of recipient cell, then grows into transgenic animals, this method is called microinjection. It is very popular and available, now many kinds of transgenic animals have been produced, such as transgenic mouse, rabbit, sheep, pig, cattle, fish, chicken and so on. Microinjection is very important to construct animal model of many diseases, and it is good for studying the pathogenesis and therapeutics of many diseases.Dopppl (Dpl, i.e. double in German) was first identified by Moore et al. and Li et al. Although it was not realized for quite some time, Dpl was first produced accidentally by generating PrPC knockout mice. To elucidate the physiological function of PrPC, several mouse lines with targeted disruptions of Prnp were independently generated. All mutant mouse lines lacked significant regions of the Prnp ORF and did not produce PrPC protein, but showed two strikingly different phenotypes. Zrch I Prnp?/? (also known as Zrch I Prnp0/0) and Edbg Prnp?/? mutant mice (named after their cities of origin, Zurich and Edinburgh) showed only minor defects, whereas Ngsk Prnp?/?, Zürich II and Rcm0 mice developed cerebellar Purkinje cell degeneration that caused ataxia with advancing age. This conundrum was solved when Moore and Li realized that in the brain of ataxic, but not of healthy, Prnp-mutant mice, expression of Prnd mRNA was upregulated. An intergenic splicing event places the Dpl locus under the control of the Prnp promoter, probably owing to the deletion of the Prnp intron 2 sequence. This intergenic splicing event could also be detected at very low levels in wild-type mice, but was greatly enhanced by the absence of the intron 2 splice acceptor. Whereas the Prnp promoter is strongly expressed in neuronal cells, the Prnd promoter is not, and therefore Prnd expression from the Prnp promoter results in overproduction of Dpl in the brain. Further experiments have demonstrated an inverse correlation between the mRNA levels of Prnd and the onset of ataxia. Disease progression is accelerated by increasing Prnd levels, supporting the idea that ectopic Dpl expression, but not functional loss of PrPC, might be responsible for neuronal degeneration in ataxic Prnp-deficient mice. Subsequent studies of Dpl transgenic mice and Prnd0/0 mice confirmed that expression of Dpl in the CNS is sufficient to produce neurodegeneration and that the neurotoxic effect of Dpl is antagonized in a dose-dependent fashion by coexpression of PrP. Dpl causes loss of both Purkinje and granule cells in the cerebellum, depending on the cell type in which the protein is expressed. Dpl also produces a leukoencephalopathy characterized by axon loss and myelin degeneration. So people are interested in research of Dpl protein.Dpl is a protein which is encoded by Prnd gene. Prnd gene is located only 16 kb downstream of Prnp in the mouse genome, the short arm of 2 chromosome. Prnd possesses 3 exons and 2 introns, the unique open reading frame (ORF) located in the second exons, which encodes 179 residues. Dpl protein is attached to the cell membrane via a glycosyl phosphatidyl inositol (GPI), the 1~23 residues of N terminal is signal peptide, 153~179 participate in the formation of GPI. There are two disulfide bonds between Cys94 and Cys145, Cys108 and Cys140. The 99th and 111th residues is Asp, which form two glycosylation sites. The primary structure of Dpl protein showes ~25% identity with the C-terminal two thirds of PrPPC, but it lacks the N-terminarlly located octameric repeats and hydrophobic region present in PrPC. The NMR structure of recombinant mouse Dpl shows that the overall topology of Dpl is similar to PrPC. Dpl contains threeα-helices and two shortβ-sheet motifs, as does PrPC,α-helices hold 40%. It is sensitive to protease K as PrPC. Normal cellular PrPC protein is also attached to the cell membrane via GPI, but PrPSc which accumulates in the kytoplasm of neuron and other infected celles, is a conformationally altered isoform of PrPC. It is known that transgenic (Tg) mice without GPI expresses″mysterious″PrPC, these mice cannot develop into prion disease when infected PrPSc. It is valuable to test whether Dpl has to be on surface of cell to exert toxicity and induce cell death, so we have generated Tg mice expressing Dpl without GPI, in order to supply Tg animal models for investigating the bionomics of Dpl protein.In this study, a recombinant Dpl plasmid without GPI (pDoppel 1-155) was digested by Not I, then the objective gene was recovered and purified. The gene was introduced into male pronucleus of Balb/c mouse fertilized ovum by microinjection, it amounts to 700 fertilized ovum, the survial is about 170, they were transplanted into ampulla of uterine tube of 17 recipient mice. We got 32 mice which have been injected the Dpl 1-155 gene, in which 6 was positive by PCR; one F0 generation of Tg mouse expressing Dpl without GPI was mated with a wild-type mouse, then 21 F1 generation mouse were got, in which 10 were positive by PCR identification. Brain tissue of three F1 generation mice which were positive by PCR identification was collectd, and was identified by Western blotting, there was 17 kDa Dpl 1-155 protein expessed in the brain. Tg mice expressed Dpl 1-155 was obtained though PCR, RT-PCR and Western blotting detection.