A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.     

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.     

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.     

Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs

Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.     

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.     

Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3' ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.     

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.     

Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis

The availability of genomic sequence information and extensive bacterial artificial chromosome (BAC) libraries for both the mouse and human genomes is ushering in a new era in biological research and disease modeling. To facilitate the study of large mammalian genes in vivo, we have developed: i) a simple lambda bacteriophage-based methodology for recombining overlapping bacterial artificial chromosomes (BACs) into a single larger BAC, and ii) a new methodology for targeting "seamless" mutations into BACs. In the first method, overlapping sequence from one BAC is cloned alongside a selectable marker and placed between unique sequence arms from the terminus of the other BAC to create a targeting construct. Two rounds of recombination-based cloning are then performed. The robustness of this methodology is demonstrated herein by using it to obtain a 254 kb BAC containing the entire human androgen receptor (hAR) gene. In the second method, transient expression of three lambda bacteriophage genes to 'pop-in' a targeting cassette is followed by RecA expression from the targeting vector itself to 'pop-out' the vector backbone. This new "hybrid recombineering" method combines the strengths of the lambda bacteriophage and RecA systems, while avoiding their major weaknesses. Application of this method for introduction of a 162 CAG repeat expansion into the hAR 254kb BAC is shown. With "hybrid recombineering", we believe that the power and utility of the classical 'pop-in/pop-out' approach -- so commonly and efficiently employed in yeast for decades -- can now be achieved with BACs.     

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a ‘targeting’ vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.     

Simple and highly efficient BAC recombineering using galK selection

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.     

Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> MW005: lambda Red-mediated recombineering and copy-number induction of <Emphasis Type="Italic">oriV</Emphasis>-equipped constructs in a single host

Background  

Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans -replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV -equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a P _BAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.     

Transposon-mediated BAC transgenesis in human ES cells

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.