A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages

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大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

Towards gene therapy of sickle cell disease

Sickle cell disease (SCD) is a significant health problem in the United States, in part of Europe and in many developing countries. Progress has been made in the management of the disease but a cure is still elusive except in the rare cases where bone marrow transplant is a practical option. Hopes for a complete universal cure rest mostly on two gene therapy approaches: correcting the mutation that causes the disease, or adding a corrective gene to the genome of the patient. The former approach is extremely attractive but is in its early stages of development. One of the most important remaining obstacles for the latter approach is the control of expression of a transgene integrated at ectopic chromosomal sites. Animal models for SCD should help in the development of the gene therapy methods discussed above, as well as the pharmacological approaches that aim at ameliorating or eliminating the symptoms of SCD.     

Cre recombinase expression controlled by the hematopoietic regulatory domain of Gata-1 is erythroid-specific

Available data suggest that gene regulation by the Gata-1 Hematopoietic Regulatory Domain (Gata-1-HRD) is limited to cells derived from the erythroid lineage. This characteristic makes Gata-1-HRD a candidate for control of cre expression in conditional knock-in and knock-out models in which erythroid-specific gene expression is essential. To characterize the specificity of Gata-1 HRD regulation of cre, transgenic mice expressing improved cre recombinase (iCre) under the control of Gata-1-HRD were generated. The founders were crossbred with mice that have an inactive loxP-containing β-galactosidase gene that can be rescued by the cre recombinase. The β-galactosidase activity was detected in the marrow of this crossbred mouse, but no activity was observed in other organs. To identify the cre expressing cells in marrow, double-immunostaining of marrow sections with anti-β-galactosidase, and antibodies against various hematopoietic lineage markers or erythropoietin receptor (epor) was performed. The epor positive cells in marrow expressed β-galactosidase, but megakaryocytic precursors and nonerythroid epor-positive cells in brain and spleen did not. We conclude that when cre is under control of Gata-1-HRD, its expression/function is limited to erythroid progenitors. The knock-in and knock-out models utilizing Gata-1-HRD-iCre, can be explored for the studies of erythroid-specific gene expression.     

Modification and identification of a vector for making a large phage antibody library

Background The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. Methods scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC Ⅲ encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. Results The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. Conclusions The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.     

Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal myelination during development and remyelination following focal demyelination

In the developing spinal cord, the majority of oligodendrocytes are derived from the ventral ventricular zone. Several recent studies suggested that a small number of oligodendrocyte precursor cells (OPCs) can also be generated in the dorsal spinal cord. However, it is not clear whether these dorsal oligodendrocyte precursor cells participate in myelination and remyelination. To investigate the fate and potential function of these dorsally-derived oligodendrocytes (dOLs) in the adult spinal cord, Cre-lox genetic fate mapping in transgenic mice was employed. We used the Pax3(Cre) knock-in mouse to drive Cre expression in the entire dorsal epithelium and the Rosa26-lacZ or Z/EG reporter line to trace their spatial distribution and population dynamics in the spinal cord. The dorsal OPCs generated from the Pax3-expressing domains migrate into all regions of spinal cord and subsequently undergo terminal differentiation and axonal myelination. In response to a focal demyelination injury, a large number of newly differentiated oligodendrocytes originated from dOLs, suggesting that dOLs may provide an important source of OPCs for axonal remyelination in multiple sclerosis or spinal cord injury.     

Cre/LoxP 系统联合SV40 LTag 诱导可复性永生化大鼠皮肤成纤维细胞系的建立

 目的探索可复性永生化大鼠皮肤成纤维细胞的建立方法。方法采用胰酶消化法分离培养大鼠皮肤成纤维细胞;逆转录病毒载体（SSR69）转染成纤维细胞,筛选稳定表达SV40 LTag 的永生化成纤维细胞;用表达Cre 重组酶的腺病毒感染永生化成纤维细胞,筛选获得回复后成纤维细胞。对原代、永生化及回复后成纤维细胞,倒置显微镜下观察细胞生长状态;采用细胞生长曲线法测定细胞增殖能力。免疫荧光染色检测Vimentin 在原代及永生化成纤维细胞中的表达情况,平板克隆实验评价回复后成纤维细胞的致瘤性。结果提取了大鼠皮肤成纤维细胞,并筛选出表达SV40 LTag 的永生化成纤维细胞,二者形态无明显差异,均贴壁生长,呈长梭形或多角形,但永生化成纤维细胞体外增殖能力明显高于原代成纤维细胞（P < 0.05）,在体外培养传代>25 代。细胞免疫荧光提示两种细胞均在细胞质内表达Vimentin。回复后成纤维细胞不表达SV40 LTag,其体外增殖能力与原代细胞无明显差异（P > 0.05）,无致瘤性。结论应用Cre/LoxP系统联合SV40 LTag 可以建立可复性永生化大鼠皮肤成纤维细胞系     

Ablation of serum response factor in dopaminergic neurons exacerbates susceptibility towards MPTP-induced oxidative stress

The high susceptibility of dopaminergic (DA) neurons to cellular stress is regarded as a primary cause of Parkinson's disease. Here we investigate the role of the serum response factor (SRF), an important regulator of anti-apoptotic responses, for the survival of DA neurons in mice. We show that loss of SRF in DA neurons does not affect their viability and does not influence dopamine-dependent behaviors. However, ablation of SRF causes exacerbated sensitivity to 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), leading to significantly greater loss of DA neurons in the substantia nigra, compared with DA neurons located in the ventral tegmental area. In addition, loss of SRF decreases levels of the anti-apoptotic proteins brain-derived neurotrophic factor (BDNF) and Bcl-2, a plausible underlying cause of increased sensitivity to oxidative stress. These observations support the notion that dysfunction of the SRF-activating mitogen-associated kinase pathway may be part of Parkinson's disease etiology.     

Conditional deletion of Indian hedgehog from collagen type 2alpha1-expressing cells results in abnormal endochondral bone formation

Indian hedgehog (Ihh) is actively involved in endochondral bone formation. Although expression of Ihh is mostly restricted to pre-hypertrophic chondrocytes, the role of chondrocyte-derived Ihh in endochondral bone formation is not completely understood. To address such unresolved issues, we used the Cre/loxP approach to generate mice (Col2alpha1Cre; Ihhd/Ihhd) in which the Ihh gene was selectively ablated from collagen type II expressing cells. Mutant mice were born with the expected ratio of Mendelian inheritance, but died shortly after birth and were smaller in size, exhibiting malformed and retarded growth of limbs with severe skeletal deformities. Alizarin red S staining showed abnormal mineralization of axial and appendicular bones. Histological analysis of mutant long bones revealed abnormal endochondral bone formation with loss of a normal growth plate. In addition, in vivo bromo-deoxyuridine (BrdU) labelling showed a marked decrease in chondrocyte proliferation. A delay in chondrocyte hypertrophy in Col2alpha1Cre; Ihhd/Ihhd mice was detected by the expression of collagen type X and osteopontin, using in situ hybridization. Furthermore, there was no expression of bone markers such as collagen type I, bone Gla protein, Runx2/Cbfa1 or PTH-R in the perichondrium of mutant mice, indicating the absence of osteoblasts from endochondral bones. Thus, selective loss of chondrocyte-derived Ihh recapitulated the defects in Ihh(-/-) animals, providing direct in vivo evidence that Ihh not only regulates chondrocyte proliferation and differentiation but also exerts effects on osteoblast differentiation. Understanding the exact functions of the molecules involved in endochondral bone formation will form the basis for further study to determine the molecular mechanisms of skeletal diseases involving various cellular components of bone.