Collagen1α1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlcd （Collα1） promoter （Collα1-Cre）. Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Coll αl-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles （Smad4^Co/Co）. Multiple tissue PCR of Collα1-Cre;Smad4^Co/＋ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Collα1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Collα1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

Inducing agent tamoxifen of CreER^(T2) to reduce the side effects of gene therapy for Parkinson’s disease

BACKGROUND：Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products.OBJECTIVE：Here we developed recombinant adeno-associated virus （AAV） as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor （CreERT2） ＋ inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine.DESIGN, TIME AND SETTING：A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004.METHODS：To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson＇s disease, with inducing agent tamoxifen to regulate gene expression.MAIN OUTCOME MEASURES：The levels of dopamine and aromatic L-amino acid decarboxylase （AADC） activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson＇s disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson＇s rats.RESULTS：Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson＇s rat striatum （P 〈 0.01） and improve the rotational behavior of Parkinson＇s rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors （P 〈 0.01）, but did not affect the expression and activity of AADC.CONCLUSION：The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2〉, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.

Modification of Cre Gene by PCR Method

Cre/LoxP site-specified recombination system is mainly used for excision,inversion and integration of target gene.Therefore,this system can be used for plant marker free genetic transformation,site-specific transgene expression and so on.However,the application of this system was limited due to its low expression and excision efficiency.In this study,an intron,which can enhance gene expression in plants,was inserted into Cre by using PCR method.And a modified Cre gene,named Crein,was obtained.This gene was ...

Expression Patterns of Inducible Cre Recombinase Driven by Differential Astrocyte-Specific Promoters in Transgenic Mouse Lines

Astrocytes are the most abundant cell type in the central nervous system(CNS).They provide trophic support for neurons,modulate synaptic transmission and plasticity,and contribute to neuronal dysfunction.Many transgenic mouse lines have been generated to obtain astrocyte-specific expression of inducible Cre recombinase for functional studies;however,the expression patterns of inducible Cre recombinase in these lines have not been systematically characterized.We generated a new astrocyte-specific Aldh1 l1-CreER^(T2)knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines(hGfap-CreER^(T2)from The Jackson Laboratory and The Mutant Mouse Resource and Research Center,Glast-CreER^(T2),Cx30-CreER^(T2),and Fgfr3-iCreER^(T2))by crossing with Ai14 mice,which express tdTomato fluorescence following Cre-mediated recombination.In adult Aldh1 l1-CreER^(T2):Ai 14 transgenic mice,tdTomato was detected throughout the CNS,and five novel morphologicallydefined types of astrocyte were described.Among the six evaluated lines,the specificity of Cre-mediated recombination was highest when driven by Aldh1 l1 and lowest when driven by hGfap;in the latter mice,co-staining between tdTomato and NeuN was observed in the hippocampus and cortex.Notably,evident leakage was noted in Fgfr3-iCreER^(T2)mice,and the expression level of tdTomato was low in the thalamus when Cre recombinase expression was driven by Glast and in the capsular part of the central amygdaloid nucleus when driven by Cx30.Furthermore,tdTomato was clearly expressed in peripheral organs in four of the lines.Our results emphasize that the astrocyte-specific CreER^(T2)transgenic lines used in functional studies should be carefully selected.

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H^＋-, K^＋-ATPase gene （Atp4b）. In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles （Smad4Ca/Co）. Multiple-tissue PCR of Atp4b-Cre;Smad4Co/＋ mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.

Generation of conditional knockout alleles for PRL-3

Phosphatase of regenerating liver-3 （PRL-3） is a member of the protein tyrosine phosphatase （PTP） superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor metastasis, we applied a rapid and efficient method for generating PRL-3 floxed mice and investigated its phenotypes. A BAC retrieval strategy was applied to construct the PRL-3 conditional gene-targeting vector. Exon 4 was selected for deletion to generate a nonfunctional prematurely terminated short peptide as it will cause a frame-shift mutation. Conditional knockout PRL-3 mice were generated by using the Cre-loxP system and were validated by Southern blot and RT-PCR analysis. Further analysis revealed the phenotype characteristics of PRL-3 knockout mice and wildtype mice. In this study, we successfully constructed the PRL-3 conditional knockout mice, which will be helpful to clarify the roles of PRL-3 and the mechanisms in tumor metastasis.