Expression Patterns of Inducible Cre Recombinase Driven by Differential Astrocyte-Specific Promoters in Transgenic Mouse Lines / 神经科学通报·英文版

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 Aldh1l1-CreER knock-in mouse line and compared the expression pattern of Cre recombinase between this and five widely-used transgenic lines (hGfap-CreER from The Jackson Laboratory and The Mutant Mouse Resource and Research Center, Glast-CreER, Cx30-CreER, and Fgfr3-iCreER) by crossing with Ai14 mice, which express tdTomato fluorescence following Cre-mediated recombination. In adult Aldh1l1-CreER:Ai14 transgenic mice, tdTomato was detected throughout the CNS, and five novel morphologically-defined types of astrocyte were described. Among the six evaluated lines, the specificity of Cre-mediated recombination was highest when driven by Aldh1l1 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 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 transgenic lines used in functional studies should be carefully selected.

Astrocyte Specificity and Coverage of hGFAP-CreERT2 Tg(GFAP-Cre/ERT2)13Kdmc Mouse Line in Various Brain Regions

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.

AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.

The Specificity and Efficiency of YFP Labeled NK Cells through Vav-Cre Induced YFP Reporter System in Mice / 天津医药

Objective To explore the specificity and efficiency of YFP labeled natural killer (NK) cells through Vav-Cre induced YFP reporter system in mice. Methods ROSA26R-YFP and Vav-Cre mice were crossed, and their YFP and Cre gene double positive progeny were screened by genotyping. The specificity of YFP in hematopoietic cells from im-mune organs including lymph nodes, spleen, thymus and bone marrow were analyzed by flow cytometry. The percentages of YFP positive cells in NK cells from lymph nodes, spleen and bone marrow were also analyzed by flow cytometry. Results A total of 11 double positive mice (ROSA26R-YFP-(+/-)VavCre) were obtained in 17 mouse offspring by crossing ROSA26R-YFP mice with Vav-Cre mice. The percentages of YFP positive cells in immune organs including lymph nodes, spleen, thy-mus and bone marrow were 73.87%± 1.51%, 56.07%± 1.47%, 86.17%± 1.74%and 53.60%± 3.56%, and there were signifi-cant differences compared with the corresponding negative control cells(0.27%±0.01%, 1.33%±0.91%, 0.11%±0.01%and 0.29%± 0.03%, P0.05). The positive rates of YFP were significantly higher in NK cells in lymph nodes, spleen and bone marrow (76.94%±0.84%、81.66%±1.18%and 88.92%±0.77%) compared with those of control (P<0.01). Conclusion YFP marked NK cells through Vav-Cre induced YFP reporter system in mice have high specificity and efficiency.

Generation of islet β cell-specific Cre recombinase targeting vector by homologous recombination in bacteria / 第二军医大学学报

Objective To construct a knock-in targeting vector for expressing of Cre recombinase specifically in islet β cell and provide the key material for knock-in mice with Crr recombinase expressed in islet β cells, providing a knock-out animal model for studying the function of islet β cells. Methods In our study, we constructed a knock-in targeting vector using the third exon of insulin 2 (Ins2) as a target site with λ phage Red recombination system. Through a first homologous recombination, we cloned an about 12 kb genomic DNA fragment from the bacterial artificial chromosome (BAC) which contained Ins2 genomic DNA into a low copy vector pBR322-2s through gap repair. Meantime, a mini-targeting vector containing internal ribosome entry site (.IRES), DNA sequences encoding Crr recombinase and positive-negative-selection (PNS) gene was generated. After second recombination, the final Ins2-Cre targeting vector was generated. Results With the third exon of Ins2 used as the target, we successfully constructed the knock-in targeting vector expressing Cre recombinase which was controlled by endogenous Ins2 gene. Conclusion We have successfully constructed the knock-in targeting vector expressing Cre recombinase, it will provide an important material forcreating animal model of Cre recombinase which is specially expressed in islet β cells.

Development of thymic lymphomas in mice disrupted of Brca2 allele in the thymus

Germ-line mutations in BRCA2 predispose to early-onset cancer. Homozygous mutant mouse, which has Brca2 truncated in exon 11 exhibit paradoxic occurrence of growth retardation and development of thymic lymphomas. However, due to its large embryonic lethality, cohort studies on the thymic lymphomas were not feasible. With the aid of Cre-loxP system, we demonstrate here that thymus-specific disruption of Brca2 allele without crossing it to p53-mutant background leads to the development of thymic lymphomas. Varying from 16 weeks to 66 weeks after birth, 25% of mice disrupted of Brca2 in the thymus died of thymic lymphomas, whereas previous report did not observe lymphomagenesis using similar Cre-loxP system. Future analysis of thymic lymphomas from these mice presented here will provide information on the cooperative mutations that are required for the BRCA2-associated pathogenesis of cancer.

Cre-loxP recombination vectors for promoter studies

For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.

Construction and application of a mouse HPRT targeting vector with Cre recombinase recognition site lox66 / 第二军医大学学报

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.

Establishment of large phage antibody library / 中国免疫学杂志

Objective:To create a large human phage antibody library from which easy to get diabody and select human antibody clones.Methods:VL and VH genes were amplified by RT-PCR from RNA which came from normal adult peripheral blood and new-born cord blood lymphocytes. VL and VH genes were reamplified to add a region of overlap in the ScFv linker to form ScFv genes in which VH genes were flanked by two non-homologous loxp sites. The ScFv genes were cloned into PDF to obtain a primary library. This primary library was used to infect bacteria Bs1365(expressing cre recombinase) with high multiplicity of infection(MOI) 200∶1. This procedure resulted in a very large phage antibody library through VL and VH recombined by the cre recombinase. Following recombination, phagemide were derived from these bacteria and used to infect bacteria not expressing cre(XL1-Blue) at MOI

Establishment of double reporter transgenic mice for monitoring Cre recombinase activity / 第三军医大学学报

Objective To establish a double reporter transgenic mice for monitoring Cre recombinase activity. Methods ZAP DNA fragment with lacZ and human alkaline phosphatase (hAP) gene was microinjected into the male pronucleus of 554 fertilized eggs from C57BL/6 mice. The founder mice and their progeny were screened for integration of transgene into the mouse genome by PCR and Southern blotting. The expression of lacZ transgene at early embryos from F1 generation mice was analyzed by X gal staining. Results A total of 398 survival ZAP DNA injected fertilized eggs were transfered to the oviducts of 21 pseudopregnant recipient mice. Of the 21 recipient mice, 13 became pregnancy and gave birth to 68 offspring mice. The zygote survival rate and birth rate were 71% (398/554) and 17% (68/398), respectively. Of the 68 offspring mice, 9 mice (5 males and 4 females) were identified by PCR and Southern blot analysis. Total integration rate and efficiency of transgene was 13% (9/68) and 1.6% (9/554), respectively. Nine mice as the founders were back crossed to set up F1 generation with other inbred C57BL/6 mice. Out of 9 transgenic mice, transmission of reporter gene in F1 offspring mice followed Mendelian rules, but the expression of lacZ protein was detected at the early embryonic stage (13.5 days postcoitum) in only 3 mice. Conclusion A double reporter transgenic mice for monitoring Cre recombinase activity is established.