Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1

The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ～1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.     

A novel model to characterize structure and function of BRCA1

BRCA1 plays a central role in DNA repair. Although N‐terminal RING and C‐terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B‐lymphocyte cell line DT40. The combination of “homologous recombineering” and “RT‐cassette” enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock‐in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2–9 BRCA1^{Δ/Δ181‐1415} (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1^{Δ/Δ126‐136} decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1^{Δ/Δ525‐881} within DNA‐binding region, even the conserved 7‐amino acid deletion BRCA1^{Δ/Δ872‐878} within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage‐induced Rad 51 foci formation in human BRCA1 knock‐in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.     

重组工程及其应用

随着功能基因组研究的需要 ,新近建立起一项新型高效的基于体内同源重组的遗传工程技术———重组工程技术。重组工程可定义为 :基于噬菌体短同源序列重组功能的遗传工程 ,或者基于同源重组的遗传工程。λ噬菌体Red系统完全不同于传统的依赖RecA的大肠杆菌重组系统 ,特点是使用长度仅为 <5 0个碱基的同源臂高效率地催化体内同源重组反应。体内重组过程不再需要预先构建含有同源序列的质粒或噬菌体的中间产物 ,只需要简单在体外合成寡核苷酸同源序列 ,或者用PCR方法合成线性打靶序列。重组反应不依赖大肠杆菌RecA系统 ,不需要限制性内切核酸酶和连接酶 ,不需要复杂的体外重组操作 ,可在大肠杆菌体内对染色体DNA、对BAC和PAC质粒或普通质粒载体进行精确的修饰 ,包括真核或原核细胞基因组DNA的基因敲除、基因敲入、基因克隆和各种突变体的引入。由于该技术具有高效率、简单性和应用的广泛性等独特优点 ,将来完全有可能取代传统的遗传工程技术。主要介绍了λ噬菌体Red重组酶系统及重组工程在功能基因组研究方面的应用与进展     

大肠杆菌重组工程

源于噬菌体的大肠杆菌同源重组系统不需要限制性内切酶和DNA连接酶就可以进行DNA克隆和亚克隆,还能快速地改造质粒、细菌人工染色体及细菌基因组染色体,是基因工程技术的一大突破,被称为重组基因工程或重组工程。该技术操作简单,效率较高,可望为功能基因组学研究提供一个有力的工具。     

Red/ET同源重组技术及其在微生物基因组挖掘中的应用进展

Red/ET同源重组技术(Red/ET recombineering)是由来源于大肠杆菌λ噬菌体的蛋白对Redα/Redβ或来源于Rac原噬菌体的蛋白对Rec E/Rec T所介导的基于短同源臂(40–50 bp)的同源重组技术,能对宿主DNA序列进行快速、高效、精确的修饰和操作。本文主要综述了2010年以来Red/ET同源重组技术在大肠杆菌及其他细菌中的研究进展,同时简要介绍了该技术在微生物基因组挖掘,尤其是在微生物基因簇的异源表达领域的应用进展。     

应用Red重组工程技术建立asd基因缺失的大肠杆菌DH10B菌株

目的:建立一株新遗传表型的大肠杆菌DH10BΔasd菌株。方法:应用pKD46介导的重组系统、kan/kil选择反选择系统、双链线性DNA重组技术和重叠引物介导的DNA重组技术,在菌株DH10B体内,对其染色体上的asd基因进行了基因敲除。结果:建立了一株二氨基庚二酸(DAP)营养缺陷型重组大肠杆菌DH10BΔasd。结论:为进一步建立以大肠杆菌DH10B为载体的DNA疫苗或基因治疗载体奠定了基础。     

Competing RNA secondary structures are required for mutually exclusive splicing of the Dscam exon 6 cluster

Alternative splicing of eukaryotic pre-mRNAs is an important mechanism for generating proteome diversity and regulating gene expression. The Drosophila melanogaster Down Syndrome Cell Adhesion Molecule (Dscam) gene is an extreme example of mutually exclusive splicing. Dscam contains 95 alternatively spliced exons that potentially encode 38,016 distinct mRNA and protein isoforms. We previously identified two sets of conserved sequence elements, the docking site and selector sequences in the Dscam exon 6 cluster, which contains 48 mutually exclusive exons. These elements were proposed to engage in competing RNA secondary structures required for mutually exclusive splicing, though this model has not yet been experimentally tested. Here we describe a new system that allowed us to demonstrate that the docking site and selector sequences are indeed required for exon 6 mutually exclusive splicing and that the strength of these RNA structures determines the frequency of exon 6 inclusion. We also show that the function of the docking site has been conserved for ~500 million years of evolution. This work demonstrates that conserved intronic sequences play a functional role in mutually exclusive splicing of the Dscam exon 6 cluster.     

一种可转移的重组工程系统 pYM-Red 的建立

重组工程是近几年发展的新型遗传工程技术. 以 PCR 扩增的线性低拷贝质粒 pACYC184 为载体,用 Gap-repair 方法从大肠杆菌 DY330 染色体上直接体内亚克隆了包括 Red 重组酶基因在内的长约 6.7 kb 的基因序列,构建了 pYM-Red 重组质粒. 在宿主菌 W3110 体内进行了染色体上 galk 基因的敲除,验证了 Red 重组酶的生物功能,并确定了影响 pYM-Red 重组效率的诱导时间和线性 DNA 片段用量. 在 42℃诱导 10 min 和线性 DNA 打靶分子浓度为 300 ng 时, pYM-Red 的重组效率可达到大约每 4 000 个电转存活细胞中有 1 个重组阳性克隆,分别比 pKD46 和 pBR322-Red 系统高 5~6 倍.