Transformation of Escherichia coli with large DNA molecules by electroporation.

We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e. molecules > 100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA. Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested.     

Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells

Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models. Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines. Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes. BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency. The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results. We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility.     

Combination of overlapping bacterial artificial chromosomes by a two-step recombinogenic engineering method

Recombinogenic engineering or recombineering is a powerful new method to engineer DNA without the need for restriction enzymes or ligases. We report here a general method for using recombineering to combine overlapping bacterial artificial chromosomes (BACs) to build larger, unified BACs. In order to test the feasibility of using recombineering to combine two large DNA fragments (>20 kb), we constructed a unified BAC containing the full-length tyrosinase-related protein-1 (Tyrp-1) gene from two library-derived BACs, one containing the 5′ regulatory elements and the other containing the 3′ coding exons. This was achieved using a two-step homologous recombination method enabled by the bacteriophage λ Red proteins. In the first step, retrieval, a large DNA fragment (~22 kb) was retrieved from one of the original BACs. In the second step, recombination, the retrieved DNA fragment was inserted into the second original BAC to form the unified BAC containing all the desired Tyrp-1 sequence. To further demonstrate the general applicability of our approach, an additional DNA fragment (~20 kb) was inserted into the unified BAC downstream of the coding region. This method should prove very useful for enabling BAC manipulation in a variety of scenarios.     

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.     

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.     

Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus

We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.     

A multi-step strategy for BAC recombineering of large DNA fragments

Recombineering techniques have been developed to modify bacterial artificial chromosomes (BACs) via bacterial homologous recombination systems, simplifying the molecular manipulations of large DNA constructs. However, precise modifications of a DNA fragment larger than 2-3 kb by recombineering remain a difficult task, due to technical limitations in PCR amplification and purification of large DNA fragments. Here, we describe a new recombineering strategy for the replacement of large DNA fragments using the commonly utilized phage/Red recombination host system. This approach involved the introduction of rare restriction enzyme sites and positive selection markers into the ends of a large DNA fragment, followed by its release from the donor BAC construct and integration into an acceptor BAC. We have successfully employed this method to precisely swap a number of large DNA fragments ranging from 6 to 40 kb between two BAC constructs. Our results demonstrated that this new strategy was highly effective in the manipulations of large genomic DNA fragments and therefore should advance the conventional BAC recombineering technology to the next level.     

鸡马立克氏病病毒814株细菌人工染色体的构建

为构建全基因组鸡马立克氏病病毒814株感染性细菌人工染色体(bacterial artificial chromosome, BAC), 首先通过构建表达Eco-gpt(xanthine-guanine phosphoribosyl transferase, XGPRT, gpt)的哺乳动物细胞基因转移遗传选择标记(1.3 kb)和带有细菌人工染色体的基本功能基因序列的鸡马立克氏病病毒重组病毒转移载体pUAB-gpt-BAC11, 将重组病毒转移载体与鸡马立克氏病病毒细胞总DNA共转染鸡胚成纤维细胞, 在选择培养基中经过8轮加压筛选, 获得并纯化重组病毒; 将重组病毒细胞总DNA电转化大肠杆菌, 筛选共获得38个BAC分子克隆化病毒, 提取BAC-DNA转染鸡胚成纤维细胞以拯救重组病毒。结果表明, MDV-BAC2 DNA再次启动病毒感染, 拯救了重组鸡马立克氏病病毒。成功构建了鸡马立克氏病病毒814株基因组全长感染性细菌人工染色体, 为方便利用现代RED/ET基因重组系统对病毒进行反向遗传操作提供了技术平台; 同时为研究鸡马立克氏病病毒的基因功能和开发新型马立克氏病疫苗奠定了基础。     

Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.     

Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.     

BAC contig development by fingerprint analysis in soybean.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.     

A genomic BAC library and a new BAC-GFP vector to study the holocentric pest Spodoptera frugiperda

Two genomic tools for the study of Lepidoptera and the holocentric structure of their chromosomes are presented in this paper. A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA partially digested with HindIII from eggs of Spodoptera frugiperda. The library contains a total of 36,864 clones with an average insert size of 125 kb, which corresponds to approximately 11.5 genome equivalents. Hybridization screening of the library was performed with eight single-copy genes, giving an average hit of 10 clones per marker gene. Colinearity between the genome and BACs was demonstrated at the triose phosphate isomerase (tpi) locus. Probing of the library with a PCR fragment internal to the 18S ribosomal gene allowed an estimation of the rDNA locus size close to 115 repeats per haploid genome. A new vector (pBAC3.6eGFP) for transient transfection into S. frugiperda cell lines has been constructed. It is based on the BAC vector, pBAC3.6e, in which a gene encoding GFP was inserted under the control of the densovirus P9 promoter. This vector has the advantage to accommodate large genomic inserts and to be transfected in a large lepidopteran host range. It was used to construct a second BAC library from Sf9 cell nuclear DNA in order to allow a comparison between somatic and cell line genome organization.     

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

BAC trimming: minimizing clone overlaps

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.     

An improved method for generating BAC DNA suitable for FISH

Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.     

Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) systems were previously developed for cloning of very large eukaryotic DNA fragments in bacteria. We report the feasibility of cloning very large fragments of eukaryotic DNA in bacteria using conventional plasmid-based vectors. One conventional plasmid vector (pGEM11), one conventional binary plasmid vector (pSLJ1711) and one conventional binary cosmid vector (pCLD04541) were investigated using the widely used BAC (pBeloBAC11 and pECBAC1) and BIBAC (BIBAC2) vectors as controls. The plasmid vector pGEM11 yielded clones ranging in insert sizes from 40 to 100 kb, whereas the two binary vectors pCLD04541 and pSLJ1711 yielded clones ranging in insert sizes from 40 to 310 kb. Analysis of the pCLD04541 and pSLJ1711 clones indicated that they had insert sizes and stabilities similar to the BACs and BIBACs. Our findings indicate that conventional plasmid-based vectors are capable of cloning and stably maintaining DNA fragments as large as BACs and PACs in bacteria. These results suggest that many existing plasmid-based vectors, including plant and animal transformation and expression binary vectors, could be directly used for cloning of very large eukaryotic DNA fragments. The pCLD04541 and pSLJ1711 clones were shown to be present at at least 4-5 copies/cell. The high stability of these clones indicates that stability of clones does not seem contingent on single-copy status. The insert sizes and the copy numbers of the pCLD04541 and pSLJ1711 clones indicate that Escherichia coli can stably maintain at least 1200 kb of foreign DNA per cell. These results provide a new conceptual and theoretical basis for development of improved and new vectors for large DNA fragment cloning and transformation. According to this discovery, we have established a system for large DNA fragment cloning in bacteria using the two binary vectors, with which several very large-insert DNA libraries have been developed.     

Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.     

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.     

Cloning a calcium channel alpha2delta-3 subunit gene from a putative tumor suppressor gene region at chromosome 3p21.1 in conventional renal cell carcinoma

We have identified loss of heterozygosity (LOH) of approx. 1 cM region around locus D3S1289 at chromosome 3p21.1 in a conventional renal cell carcinoma (RCC). During construction of a YAC/BAC contig for this region and shotgun sequencing of BACs 277p5, 55m24 and 428i24, we detected four new microsatellites. We narrowed down the target region by analysing these new loci to less than 100 kb within the BAC 55m24 and subsequently cloned a human calcium channel alpha2delta-3 subunit gene. This gene is widely expressed in fetal tissues and different types of adult tumors. The exons of the alpha2delta-3 subunit gene are distributed along approx. 500 kb DNA sequences. As the LOH involved exclusively intronic sequences and sequencing the entire coding region did not reveal any mutation, the alpha2delta-3 subunit gene is probably not a tumor suppressor gene.