Expression of Nkx2-5-GFP bacterial artificial chromosome transgenic mice closely resembles endogenous Nkx2-5 gene activity

Mouse Nkx2-5 gene is essential for early heart development and it is regulated by a complex array of regulatory modules. In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone. We employed the recombination genes redalpha and redbeta under the pBAD promoter, which was specifically induced by the addition of L-arabinose. Recombination was selected for by our universal targeting cassette which conferred kanamycin resistance in bacterial cells and neomycin resistance in mammalian cells. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. BAC transgenics using the GFP reporter offers an efficient model system to study gene expression and regulation.     

HCV core protein-expressing DNA vaccine induces a strong class I-binding peptide DTH response in mice

In previous studies, we developed mouse genetic models and discovered genetic components of quantitative trait loci on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and fracture healing. However, these regions contain dozens of genes in several overlapping bacterial artificial chromosomes (BACs) and are difficult to clone by physical cloning strategies. A feasible and efficient approach of identifying candidate genes is to transfer the genomic loci in BAC clones into mammalian cells for functional studies. In this study, we retrofitted a BAC construct into herpes simplex virus-1 amplicon and packaged it into an infectious BAC (iBAC) to test gene function in a cell-based system, using a 128-kb clone containing the complete bone morphogenetic protein-2 (BMP-2) gene. We transduced MC3T3-E1 cells with the iBAC bearing BMP-2 gene and examined transgene expression and function. Our results have demonstrated that an iBAC can efficiently deliver a BMP-2 genomic locus into preosteoblast cells and express functional BMP-2 protein, inducing a phenotype of cell differentiation, as indicated by an increase in alkaline phosphatase activity. Therefore, this experimental system provides a rapid, efficient cell-based model of high-throughput phenotypic screening to identify the BAC clones from physically mapped regions that are important for osteoblast differentiation. It also illustrates the potential of iBAC technology in functional testing of single nucleotide polymorphisms located in the distal promoter or/and intron regions responsible for low bone density.     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette

There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human beta-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants.     

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.     

A 178-kb BAC transgene imprints the mouse Gtl2 gene and localizes tissue-specific regulatory elements

The regulation of genomic imprinting, the allele-specific expression of an autosomal gene, is complex and poorly understood. Imprinted genes are organized in clusters, where cis-acting regulatory elements are believed to interact to control multiple genes. We have used BAC transgenesis in the mouse to begin to delineate the region of DNA required for proper expression and imprinting of the mouse Delta-like1 (Dlk1) and Gene-trap locus2 (Gtl2) imprinted genes. We demonstrate that the Gtl2 gene is expressed from a BAC transgene in mouse embryo and placenta only upon maternal inheritance, as is the endogenous Gtl2 gene. Gtl2 is therefore properly imprinted on the BAC in an ectopic chromosomal location and must carry with it all necessary imprinting regulatory elements. Furthermore, we show that the BAC Gtl2 gene is expressed at levels approaching those of the endogenous gene only in the brain of adult animals, not in other sites of endogenous expression such as the pituitary, adrenal, and skeletal muscle. These data localize the enhancer(s) for brain Gtl2 expression, but not those for other tissues, to the DNA contained within the BAC clone. As the Dlk1 gene is not expressed from the BAC in any tissues, it must require additional elements that are different from those necessary for Gtl2 expression. Our data refine the interval for future investigation of Gtl2 imprinting and provide evidence for distinct regulation of the linked Dlk1 and Gtl2 genes.     

连续3步缺口修复构建小鼠乳清酸蛋白-人溶菌酶杂合基因座

目的:构建一个利用小鼠乳清酸蛋白(mWAP)基因座完整的上下游调控序列指导人溶菌酶(hLYZ)基因组序列在乳腺内特异性高效表达的mWAP-hLYZ杂合基因座,实现人溶菌酶的高效表达。方法:采用连续3步缺口修复的方法。首先,以pBR322载体作为骨架,插入预先合成的6个同源臂序列,构成能够连续进行3次缺口修复的基因抓捕载体。然后在大肠杆菌内利用λ噬菌体Red同源重组系统介导的同源重组方法:第一步,从含mWAP基因座的细菌人工染色体(BAC)上亚克隆8 kb的mWAP基因3’端完整侧翼序列到抓捕载体上;第二步,从含hLYZ基因的BAC上亚克隆5 kb的从起始密码子(ATG)到终止密码子(TAA)的hLYZ基因组序列;第三步,从mWAP BAC上亚克隆9kb的mWAP基因5’端完整侧翼序列,并使上述3个片段在抓捕载体上自动无痕地连接在一起。结果:构建了全长约22 kb的mWAP-hLYZ杂合基因座,经PCR扩增、限制性内切酶酶切和序列测定验证,构建的杂合基因座达到原mWAP基因座中mWAP基因组编码序列从起始密码子(ATG)到终止密码子(TAA)完全被hLYZ基因组序列精确置换的目的。结论:通过连续3步缺口修复构建杂合mWAP-hLYZ基因座乳腺表达载体,为乳腺生物反应器高效表达人溶菌酶提供了可行的思路和方法。     

连续3步缺口修复构建人β肌动蛋白-人凝血因子Ⅶ杂合基因座

目的:为了获得稳定高效表达凝血因子Ⅶ的哺乳动物细胞株,构建一个利用人β肌动蛋白(hACTB)基因座完整的上下游调控序列指导人凝血因子Ⅶ(hFⅦ)基因组序列在人胚胎肾细胞特异性高效表达的hACTB-hFⅦ杂合基因座。方法:采用3步连续缺口修复的方法。首先,以pBR322载体作为骨架,插入预先合成的6个同源臂,构成能进行3次连续基因抓捕的载体。然后在大肠杆菌内利用Red同源重组系统介导的缺口修复技术:第一步,从含hACTB基因座的细菌人工染色体(BAC)上亚克隆10 kb的hACTB基因3′端完整侧翼序列;第二步,从hFⅦBAC上亚克隆13 kb的从起始密码子(ATG)到终止密码子(TAG)的hFⅦ基因组序列;第三步,从hACTB BAC上亚克隆20kb的hACTB基因5′端完整侧翼序列,并使这3个基因片段自动无痕地连接在基因抓捕载体上,形成全长约50 kb的hACTB-hFⅦ杂合基因座。结果:经过PCR扩增、限制性内切酶消化和序列测定验证,构建的杂合基因座达到了原来hACTB基因座中hACTB基因组编码序列从起始密码子到终止密码子被hFⅦ从起始密码子到终止密码子的基因组序列基因组序列精确置换的目的。结论:连续3步缺口修复构建杂合基因座细胞表达载体的技术,将为细胞高效表达大载体的制备提供一种全新的思路和方法。     

An infectious transfer and expression system for genomic DNA loci in human and mouse cells.

The recent completion of the human genome sequence allows genomics research to focus on understanding gene complexity, expression, and regulation. However, the routine-use genomic DNA expression systems required to investigate these phenomena are not well developed. Bacterial artificial chromosomes (BACs) and P1-based artificial chromosomes (PACs) have proved excellent tools for the human genome sequencing projects. We describe a system to rapidly and efficiently deliver and express BAC and PAC library clones in human and mouse cells by converting them into infectious amplicon vectors. We show packaging and intact delivery of genomic inserts of >100 kilobases with efficiencies of up to 100%. To demonstrate that genomic loci transferred in this way are functional, the complete human hypoxanthine phosphoribosyltransferase (HPRT) locus contained within a 115-kilobase BAC insert was shown to be expressed when delivered by infection into both a human HPRT-deficient fibroblast cell line and a mouse primary hepatocyte culture derived from Hprt-/- mice. Efficient gene delivery to primary cells is especially important, as these cells cannot be expanded using antibiotic selection. This work is the first demonstration of infectious delivery and expression of genomic DNA sequences of >100 kilobases, a technique that may prove useful for analyzing gene expression from the human genome.     

Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene β-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome （BAC） libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR （qPCR） revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process.     

A general method to modify BACs to generate large recombinant DNA fragments

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA frament containing the inverted human α-globin genes (ϑ, α1, α2, and ζ) from BAC191K2 and the locus control region (LCR) of human β-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments. These two authors contributed equally to this work.