Bacterial Artificial Chromosome Construct Research Articles

Genomic and cytological analysis of the Y chromosome of Drosophila melanogaster: telomere-derived sequences at internal regions

The genomic analysis of heterochromatin is essential for studying chromosome behavior as well as for understanding chromosome evolution. The Y chromosome of Drosophila melanogaster is entirely heterochromatic and the under-representation of this chromosome in genomic libraries together with the difficulty of assembling its sequence has made its study very difficult. Here, we present the construction of bacterial artificial chromosome (BAC) contigs from regions h14, h16 and the centromeric region h18. The analysis of these contigs shows that telomere-derived sequences are present at internal regions. In addition, immunostaining of prometaphase chromosomes with an antibody to the kinetochore-specific protein BubR1 has revealed the presence of this protein in some Y chromosome regions rich in telomere-related sequences. Collectively, our data provide further evidence for the hypothesis that the Drosophila Y chromosomes might have evolved from supernumerary chromosomes.

LAMBDA RED-MEDIATED BAC RECOMBINEERING ALLOWS REAL-TIME VISUALIZATION OF PANCREAS DEVELOPMENT IN LIVING ZEBRAFISH EMBRYOS.

Background: The basic helix-loop-helix transcription factor ptf1a plays a central role in the mammalian pancreatic development. In zebrafish, its expression is observed in the developing hindbrain and pancreas in a temporally and spatially restricted pattern. To establish an in vivo system for visualizing developing pancreas, we generated a bacterial artificial chromosome (BAC) construct having green fluorescence gene under the regulation of ptf1a promoter by engineering BAC through homologous recombination. Materials and Methods: The template plasmid pKD4-GFP was constructed by cloning GFP gene into pKD4 vector. BAC CH211-142H2 (Encompassing zebrafish ptf1a locus) targeting constructs were generated by PCR. Within each primer, the first 39–41 bp was homologous to the pft1a sequence on BAC, and the next 20–21 bp was complementary to the template plasmid. Bacteria carrying both BAC and pKD46 (λ Red recombinase) were transformed with targeting constructs by electroporation. Positive colonies having undergone homologous recombination replacing ptf1a-coding sequence with GFP were selected. The purified BAC construct containing the EGFP gene under the regulation of zebrafish ptf1a promoter was injected directly into the yolk of zebrafish embryos at single-cell stage. Results: Transient GFP expression was observed in hindbrain and retina at 24 hpf and in pancreas at 32 hpf. In hindbrain, the fluorescence was gradually intensified till 48 hpf, maintained for 1–2 days, and extinguished at 96 hpf. Pancreatic fluorescence was increased till 3–4 dpf and maintained for several days. The fluorescence was confined to exocrine pancreas and was excluded from the central islet. The expression rate and intensity was dose-dependent, i.e. 25 % with 1–2 ng and 50% with 3–4 ng. GFP expression was specific and reproducible, and ectopic expression was observed in less than 10%. Those fishes with fluorescence are being raised and their F1 progeny will be screened for GFP expression. Conclusions: Developing pancreas can be visualized in living zebrafish embryos and the transgenic line, once established, will provide a useful model of labeled exocrine pancreatic cells for in vivo.

Inflammatory Bowel Disease: A Genomic Picture Predicts a Changing Response From the Laboratory–Part II

IBD-1 Linkage Analysis Hugot and colleagues1 described the first genome screen for CD in 1996. The investigators initially screened 2 consecutive and independent panels of families comprising 40 affected sibling pairs using 270 markers throughout the genome. An analysis of allele sharing by affected sibling pairs showed that only 4 markers achieved statistical significance. Using data obtained from a second set of affected families localized the susceptibility locus to a pericentric region of chromosome 16. The initial work by Hugot was greeted with concern and mild skepticism. Prior to this time, susceptibility loci for other complex diseases had rarely been replicated. Examples of unimpressive attempts to duplicate these sorts of findings can be found in multiple sclerosis, affective disorders, and type II diabetes. At the same time, success had been achieved with high penetrance Mendelian syndromes, most notably the breast cancer-1 gene (BRCA1) which had been discovered using a similar linkage analysis process. The contrasting levels of success diverge according to inheritance patterns. Mendelian inheritance offers hope for finding susceptibility loci, while non-Mendelian (ie, complex) diseases usually failed critical reanalysis. Refinements in the localization of IBD1 proceeded immediately. By employing a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping and construction of bacterial artificial chromosomes, Hugot and colleagues identified 3 independent single nucleotide polymorphisms (SNPs) associated with CD.2 A frameshift polymorphism and 2 missense variants of NOD2 were found within the LRR domain encoded by the gene. Genotyping of patients with UC did not show an association with these SNPs. This observation agrees with earlier data showing the lack of linkage between UC and the IBD1 susceptibility locus.3 A connection to immune dysregulation is readily apparent. NOD2 belongs to the Apaf-1/Ced-4 superfamily After decades of stagnation, molecular discoveries have altered our view of inflammatory bowel disease (IBD). This recent change in our understanding of the IBD’s pathogenesis combined with emerging biologic therapeutics will benefit the laboratory, the clinician, and the patient. science [chemistry | molecular diagnostics] Inflammatory Bowel Disease: A Genomic Picture Predicts a Changing Response From the Laboratory–Part II

Creating porcine biomedical models through recombineering

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide ‘clones’ of genetically modified animals.

Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size

The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the 'single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 microg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 x TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 x TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning.

Construction of a bacterial artificial chromosome library from the inshore hagfish, Eptatretus burgeri: A resource for the analysis of the agnathan genome

The jawless fish occupy an important phylogenetic position for understanding the evolution of body plans, the origin of adaptive immunity and genome evolution in chordates. We describe here the construction of a large-insert bacterial artificial chromosome (BAC) library from the inshore hagfish, Eptatretus burgeri. The BAC library contains 93,978 clones with an average insert size of 100 kb and is estimated to represent threefold genome-equivalent coverage. The library was organized in three-dimensional pools to facilitate screening by PCR. We have screened this library by PCR and isolated several BAC clones; the average number of positive clones was compatible with the estimated genome coverage of the library. This BAC library, constructed for the first time from the jawless fish, should serve as a useful resource for the scientific community.

Harvesting the Genomic Promise: Recombineering Sequences for Phenotypes

The past decade has witnessed the construction of linkage and physical maps defining quantitative trait loci (QTL) in various domesticated species. Targeted chromosomal regions are being further characterized through the construction of bacterial artificial chromosome (BAC) contigs in order to isolate and characterize genes contributing towards phenotypic variation. Whole‐genome BAC contigs are also being constructed that will serve as the tiling path for genomic sequencing. Harvesting this genetic information for biological gain requires either genetic selection or the production of genetically modified animals. This later approach when coupled with nuclear transfer technology (NT) provides “clones” of genetically modified animals. However, to date, the production of genetically modified animals has been limited to either microinjection of small gene constructs into embryos with random insertion or complex gene constructs designed to knock‐out targeted gene expression. Neither of these approaches provides for introducing directed genetic manipulation allowing for allelic substitution [knock‐in], subsequent analyses of gene expression, and cloning. An alternative approach utilizing genomic sequence information and recombineering to direct gene targeting of specific porcine BACs is presented here.

Analysis of the flanking regions of the human α-lactalbumin gene responsible for position-effect independent expression

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the α-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The −6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the −3.4 to −0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at −1.0 and −2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human α-lactalbumin gene.

Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

Large-insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.

Cloning, transcription and chromosomal localization of the porcine whey acidic protein gene and its expression in HC11 cell line

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine (Simpson et al., 1998. J. Mol. Endocrinol. 20, 27–35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.

Acid assisted consolidation of silver alloy powders : M.M.Wray et al. J. of Research of NIST, USA, vol 103, no 5, 1998, 511–516

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183–194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.

Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.

Construction of an approximately 2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2.

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artificial chromosome (YAC) physical map is described. An approximately 2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.

Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.