Exploring taxonomic diversity and biogeography of the family Nemacheilinae (Cypriniformes)

Nemacheilidae, in the superfamily Cobitoidea, is comprised of many of morphologically similar fish species that occur in Eurasian water bodies. This large group shows inconsistencies between traditional morphological taxonomy and molecular phylogenetic data. We used mitochondrial genomes, recombinase‐activating gene proteins 1 (RAG1) and the mitochondrial cytochrome c oxidase I gene (COI) to study the phylogenetic relationships among Nemacheilidae species using Bayesian inference and maximum likelihood approaches. Phylogenetic analyses based on mitogenomes provided support for two clades (I and II). The mitogenomes, RAG1, and COI results indicated that several species and genera were not consistent with the traditional morphological subdivisions. The two clades inferred from mitogenomes showed clear geographical patterns. The Tibetan Plateau, Hengduan Mountains, and the Iran Plateau may act as a barrier dividing the clades. The estimated timing of clades separation (36.05 million years ago) coincides with the first uplift of the Tibetan Plateau. We conclude that the geological history of the Tibetan Plateau played a role in the diversification and distribution of the Nemacheilidae taxa. These results provided a phylogenetic framework for future studies of this complex group.     

产L-苹果酸重组大肠杆菌的构建

基于产琥珀酸重组大肠杆菌E.coli B0013-1050的琥珀酸合成途径,利用Red同源重组技术结合Xer/dif重组系统敲除富马酸酶基因fumB、fumC,苹果酸酶基因maeB,构建L-苹果酸合成途径,最终得到重组大肠杆菌E.coli2030,该菌株在15 L发酵罐中,产L-苹果酸12.5 g/L,葡萄糖-苹果酸转化率为52.1%,同时对发酵产物中主要杂酸丙酮酸和琥珀酸的生产原因进行了初步的探讨与分析。为进一步提高L-苹果酸的转化率,整合表达来源于黄曲霉的苹果酸脱氢酶基因,构建重组菌E.coli 2040,在15 L发酵罐中产L-苹果酸14 g/L,葡萄糖-苹果酸转化率提高到60.3%。     

禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失株的构建

目的构建大肠埃希菌强毒力岛（HPI）全岛缺失突变株,为进一步评价大肠埃希菌HPI的功能打下基础。方法根据已知大肠埃希菌HPI基因序列设计PCR敲除引物,引物5′端有50 bp的拟敲除基因的同源臂,3′端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,利用pKD46的λ重组系统替换E.coli ZL基因组上的毒力岛全岛基因,再利用表达Flp重组酶的质粒pCP20,可将FRT位点之间的氯霉素抗性基因删除,用鉴定引物进行鉴定并测序。结果构建的全岛缺失株与预期一致。结论成功构建了禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失突变株。     

Synapsis in phage Bxb1 integration: selection mechanism for the correct pair of recombination sites

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定

目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。     

Cre重组酶表达载体的构建及其在转基因小鼠胰腺中的表达

组织特异性表达Cre重组酶的转基因小鼠是进行组织特异性条件敲除研究的关键。采用PCR扩增大鼠胰岛素基因705bp启动子指导发胰岛细胞中特异表达;同时采用改构的Cre重组酶基因,在其5'端添加有真核核糖体结合序列和核定位序列使Cre重组酶能穿越核膜在细胞核能发挥功能;同时,为了保证原核基因Cre能在真核系统顺利表达,在其3'端添加含内含子的人生长激素基因。构建的表达载体在去除原核序列后用显微注射方法转基因小鼠,在出生的27只仔鼠中,PCR检测共获得7只Cre整合阳性的转基因小鼠,整合率26％。这种Cre转基因小鼠与基因组小携带LoxP位点的条件基因打靶小鼠交配,在胰腺组织中可以检测到Cre介导的重组,表明Cre在转基因小鼠胰腺中有表达。     

A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.