Establishment And Identification Of Transgenic Mice Brain-specifically Expressing Calbindin-D28k

Calcium-binding protein (Calbindin D-28k, CaBP-D28k) is a member of EF-arm calcium-binding protein family with wide distribution in the nervous system. CaBP can bind with calcium ions, caspase-3, plasma membrane ATP-PDE, P38 and other substances to regulate their activities. the The substantia nigra of Parkinson's disease (PD) patients contains a small amount of the DA neurons with strong ability of anti-degeneration, the cytoplasm of which contains CaBP. In the cases of ischemic and neural excitotoxicity, CaBP-positive neurons can survive, implying that CaBP has a protective effect on nerve cells. Apoptosis is an important mechanism of neurological degenerative diseases, including PD. Intracellular calcium overload is one of the incentives of the neuronal apoptosis. As a fast calcium buffer protein, CaBP can bind to calcium ion to limit the intracellular free calcium concentration and can be used as a enzyme activator to activate Ca2+-ATP enzyme to regulate calcium ion concentration. In addition, CaBP also can regulate the ion channels to promote the spread of calcium in the nerve cells and to protect cells from calcium overload damage under the situation of high-intensity neural activity. Presently, the study on CaBP over expression in DA neurons are limited to cell transfection or using viral vectors for in vivo study. Therefore, transgenic animal models with CaBP over-expression in the DA nerve cells has not been reported and the mechanism for protecting DA neurons by CaBP needs further investigation.To construct a brain-specific expression vector, the 3650-bp mouse TH promoter was amplified by PCR and used as the control element for expression of enhanced green fluecent protein (EGFP) reporter gene. After demonstration the brain cell-specific transcription activity of the TH promoter by transfection assay, CaBP-D28k cDNA was amplified from mouse brain tissue by RT-PCR and cloned into the TH promoter-containing vector. The recombinant vector pTH-CB was transfected into MN-9D cells and CaBP-D28k over expression was detected by RT-PCR and immunoprecipitation. In 6-hydroxydopamine-induced transfected cells, expression of anti-apoptotic factor bcl-2 was detected by in-cell Western blotting. The anti-apoptotic effect of the over expressed CaBP-D28k was further investigated by Hoechst staining, caspase-3 activity and annexin V/PI detection.To generate a transgenic mouse model for PD study, the sperm cells were incubated with pTH-CB vector in the presence of 2% DMSO and the in vitro fertilized embryos were transplanted into pseudo-preganent mice. The transgenic offsprins were screened by PCR and exogenous CaBP expression in their spars compacta of substantia nigra, ventral legmental area, hippocampus and cerebral cortex by immunofluorescence or Western blotting. The anti-injury effect of the over expressed CaBP was investigated using MPTP-simulated PD model based on normal activity, swimming test, suspension experiments and changes of TH-positive cell number in SNc. The results showed that the nucleotide sequence of the PCR-amplified 3650-bp TH promoter was identical to the published sequence. In pTH-EGFP-transfected MN-9D and ECV cell cultures, EGFP-positive cells was 7.7±0.3% and 0.7±0.1%, indicating cell slecificity of the TH promoter-controled reporter gene expression. The key regulatory elements in 3650-bp TH promoter were further investigated by PCR amplification of its 450-bp,1500-bp,2000-bp and 2400-bp fragments and located in the 2400-2000bp region using the same cell transfection assay. Sequence analysis showed that the RT-PCR-amplified CaBP-D28k cDNA contained an 785-bp open reading frame, which is identical to the published sequence in GenBank. In pTH-CB-transfected MN-9D cells, the level of CaBP-D28k mRNA was significantly higher than that of the control cells. Over-expression of CaBP could significantly enhance expression of bcl-2 and inhibit caspase-3 activity in the transfected cells. Hoechst staining and annexin V/PI assay showed that CaBP over expression could significantly reduce the number of apoptotic cells. From 96 offspring mice obtained, four were PCR-positive and two of them could stably transmit the transgene with significantly higher numbers of CaBP-positive cells in SNc. In SNc and VTA of the transgenic mice, expression levels of CaBP were significantly higher than that in wild mice, without significant change in the Hi and CC. The number of TH-positive cells in the SNc and behavioral changes of transgenic mice had no significant difference in the MPTP-induced PD model, confirming that over expression of CaBP could resist to the MPTP-induced DA neuron damage in the brain.