Lambda Phage Vector Research Articles

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.

Noreen Elizabeth Murray CBE. 26 February 1935 — 12 May 2011

Noreen Murray was one of the architects of the recombinant DNA revolution that transformed the study of biology from the early 1970s. Her particular prowess for genetic manipulation of bacteria and their phage was critical in developing the bacteriophage lambda vectors that were a vital part of the early genetic engineering toolbox. Her skill as a microbial geneticist had earlier become apparent through her work on genetic recombination and complementation in the fungus Neurospora , especially as a postdoctoral researcher at Stanford where her work brought her to the attention of some of the giants of early molecular biology. Back in the UK, first at Cambridge and then, for the bulk of her career, at Edinburgh, she produced a remarkable body of work focused on uncovering the mechanisms and biology of restriction enzymes, and their adaptation as tools underpinning modern biological research and the rise of the biotechnology industry. Much of this work was done in collaboration with her husband Ken Murray FRS, whose biographical memoir accompanies this one. Together they were known not only for the quality of their research but also for their vast generosity both on a personal level and on a larger canvas through their philanthropy.

Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein

A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vector were administered intramuscularly on days 0, 14 and 28; the attenuated vaccine was given once on day 0. Half the animals in each group were challenged on day 42 by intraperitoneal injection of live C. abortus and sacrificed on day 49. Phage-vaccinated mice developed moderate antibody levels against C. abortus and yielded higher levels of IFN-γ and IL-2 compared with the attenuated live vaccine group. Clearance of chlamydiae from spleens was significantly better in the attenuated vaccine group compared with the phage vaccine group, while both groups were significantly superior to the DNA vaccine and control groups ( p < 0.01). Although levels of protection in the mouse model were lower in phage-vaccinated animals, than in 1B vaccinated animals, phage vaccines offer several other advantages, such as easier handling and safety, potentially cheaper production and no chance of reversion to virulence. Although these are preliminary results in a model system, it is possible that with further optimisation immunization with phage vaccines may provide a novel way to improve protection against C. abortus infection and trials in large animals are currently being initiated.

Can painting human cells with exogenous maltoporin enable efficient therapeutic gene transfer by bacteriophage lambda vectors?

Many gene therapy strategies require transfer of highmolecular weight DNA into human cells. To enable clinical trials, these vectors need to be produced on a large scale and at low cost. The production of effective high-capacity vectors like HSV-amplicons and helperdependent adenoviral vectors is difficult to up-scale, so new inexpensive vectors are needed for the efficient delivery of high-molecular weight DNA to human cells. Bacteriophage lambda vectors can accommodate up to about 46 kb of therapeutic DNA and can be easily produced in an industrial setting. However, the lambda vectors transfer DNA into mammalian cells with only a low efficiency. It was shown that bacteriophage lambda virions ejected their DNA in the presence of the purified receptor for bacteriophage lambda, maltoporin (LamB protein), encoded by the malB gene of Shigella sonnei 3070. This property of S. sonnei maltoporin was exploited for the bacteriophage injection-driven DNA loading of liposomes and other polymer nanocontainers displaying maltoporin. Relying on the above evidence I hypothesize that the efficient gene transfer by industrially produced bacteriophage lambda vector virions, such as cosmid transducing particles, to human cells can be accomplished after incorporation (protein painting) of the purified S. sonnei maltoporin into the human plasma membrane.

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10(5) Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K(d)) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K(d) value of 10(-7) M.

Proteasome inhibitors enhance bacteriophage lambda (λ) mediated gene transfer in mammalian cells

Bacteriophage lambda vectors can transfer their genomes into mammalian cells, resulting in expression of phage-encoded genes. However, this process is inefficient. Experiments were therefore conducted to delineate the rate limiting step(s) involved, using a phage vector that contains a mammalian luciferase reporter gene cassette. The efficiency of phage-mediated gene transfer in mammalian cells was quantitated, in the presence or absence of pharmacologic inhibitors of cell uptake and degradation pathways. Inhibitors of lysosomal proteases and proteasome inhibitors strongly enhanced phage-mediated luciferase expression, suggesting that these pathways contribute to the destruction of intracellular phage particles. In contrast, inhibition of endosome acidification had no effect on phage-mediated gene transfer, presumably because phage lambda is tolerant to extended exposure to low pH. These findings provide insights into the pathways by which phage vectors enter and transduce mammalian cells, and suggest that it may be possible to pharmacologically enhance the efficiency of phage-mediated gene transfer in mammalian cells. Finally, the data also suggest that the proteasome complex may serve as an innate defense mechanism that restricts the infection of mammalian cells by diverse viral agents.

Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells

Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcγRI, but not its associated γ-chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB, and FcγRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcγRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

In vivo gene delivery and expression by bacteriophage lambda vectors

AimsBacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo.Methods and ResultsMice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer.ConclusionsUnmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up – at least in part – by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer.Significance and Impact of the StudyThese experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage λ Q − vector

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

Analysis and Identification of Serological Tumor Antigens in Patients Treated with the GM-CSF-Gene Transduced-Autologous Tumor Vaccines (GVAX®) for Renal Cell Carcinoma.

Vaccination of GM-CSF-transduced tumor cells (GVAX®) has been demonstrated to induce cellular and humoral immune response to tumors in animal studies as well as human clinical trials. We have recently done clinical trials of GVAX® using autologous renal cell cancer (RCC) cells. Four patients with advanced RCC, who were entered to our clinical gene therapy trial, received multiple GVAX® injections every two weeks. Two out of four patients, Cases 2 and 4, are long term survivors living more than 62 and 42 months, respectively, after the start of vaccination. To examine whether the therapeutic regimen induced antitumor antibody responses in them, we compared the serum antibody reactivity against autologous tumor cell lysates before and after the vaccinations by immunoblot analysis. Using post-therapy serum as probes, proteins of around 250 kDa and 60 kDa generated clear signals, whereas the pre-vaccination serum showed no or only weak signals at the same position, suggesting that the gene therapy induced an antibody response in them. In three of them (Cases 2–4), the induction of antibodies immunoreactive with these two proteins was significant, while the results were less clear in Case 1. The suspected common antigen was actually demonstrated in both RCC lysates and normal renal cell lysates, and in human lip-derived fibroblasts, but not in H69 lung cancer cells. The strongest signal was observed in Case 2 serum obtained at 67 days after the initial vaccination, between the 5th and 6th vaccinations. This response was maintained from day 67 until day 281, just after the 17th vaccination, although it decreased slightly. After the last vaccination, the immunoreactivity remained at a lower level, although it did not disappear completely. Based on the above results, we applied SEREX, the serological identification of antigens by recombinant expression cloning, using patient's serum, to isolate the serological RCC tumor antigen genes. We constructed RCC cDNA-expression libraries on lambda phage vector, and screened the plaques with Case 2 serum pooled between days 25 and 80 after the first vaccination. The mRNA source was Case 2 RCC or VMRC-RCW RCC cell line. From above libraries, totally two million recombinant phages screened, twenty-eight SEREX positive clones, which passed secondary or further screening, were isolated. Sequencing analysis revealed the clones were composed of thirteen independent gene products, some of them have not been reported as SEREX defined antigen in previous studies. Expression profiling experiment showed, one of the newly defined SEREX antigen, testicular nuclear autoantigenic sperm protein (tNASP) gene expression had tendency to be dominantly expressed in tumor tissue compared with corresponding normal tissue. To precisely determine the serum antibody titre against the isolated SEREX defined antigens, we constructed the recombinant antigen/epitope tag expression vector and expressed in E. coli. The antibody titration in cancer patients or healty donor against these antigens using obtained recombinant antigen protein has been under investigation. The identification of the new anti-RCC antigen might cast a new light on the treatment of patients suffered from advanced RCC.

582. Detection of Enhanced Serum Antibody Production to Renal Cell Cancer Proteins and Identification of Serological Tumor Antigens in Patients Treated with the GM-CSF-Gene Transduced-Autologous Tumor Vaccines (GVAX®)

Vaccination of GM-CSF-transduced tumor cells (GVAX®) have been demonstrated to induce cellular and humoral immune response to tumors in animal studies as well as human clinical trials. We have recently done clinical trials of GVAX® using autologous renal cell cancer (RCC) cells in Japan. Four out of six advanced RCC who were entered to our clinical gene therapy trial were injected with GVAX. First of all, to examine whether the therapeutic regimen induced antitumor antibody responses in them, we compared the serum antibody reactivity against autologous tumor cell lysates before and after the vaccinations by immunoblot analysis. Using post-therapy serum as probes, high molecular weight proteins of around 250 kDa generated clear signals, whereas the pre-vaccination serum showed no or only weak signals at the same position, suggesting that the gene therapy induced an antibody response in them. In three of them (Cases 2–4), the induction of antibodies immunoreactive with the 250 kDa proteins was significant, while the results were less clear in Case 1. The suspected common antigen was actually demonstrated in both RCC lysates and normal renal cell lysates, and in human lip-derived fibroblasts, but not in H69 lung cancer cells. Furthermore, the changes in the magnitude of the antibody immunoreactivity over time were analyzed using serum from Case 2. The strongest signal was observed in serum obtained at 67 days after the initial vaccination, between the 5th and 6th vaccinations. This response was maintained from day 67 until day 281, just after the 17th vaccination, although it decreased slightly. After the last vaccination, the immunoreactivity remained at a lower level, although it did not disappear completely. Secondly, based on the results of Western blot analysis, we applied SEREX, the serological identification of antigens by recombinant expression cloning, using patient's serum. We constructed RCC cDNA-expression library on lambda phage vector, and screened the plaques with Case 2 serum pooled between days 25 and 80 after the first vaccination. The mRNA source was Case 2 autologous cultured tumor cells. Sequencing analysis of isolated positive clones (twelve in total) revealed some of them were reported as SEREX defined antigen in previous studies but others were not. Recombinant antigen proteins, fused to epitope tags, were expressed in E. coli and the antibody titration of sera from patients or healthy donner is now going on. The identification of the new anti-RCC antigen might cast a new light on the treatment of patients suffered from advanced RCC.

GENERATION AND CHARACTERIZATION OF CDNA CLONES FROM SARCOPTES SCABIEI VAR. HOMINIS FOR AN EXPRESSED SEQUENCE TAG LIBRARY: IDENTIFICATION OF HOMOLOGUES OF HOUSE DUST MITE ALLERGENS

Molecular studies on scabies, a disease of considerable human and veterinary significance, have been limited because of the difficulty of obtaining the causative organism Sarcoptes scabiei, the "itch mite." We have used skin from the bedding of crusted scabies patients as a source of mites for the construction of libraries of cDNAs from S. scabiei var. hominis in the bacteriophage lambda vector lambdaZAP express. Sequences of 145 clones established that the libraries predominantly contain sequences from S. scabiei, enabling a major sequencing program to begin. Among those sequenced to date, cDNAs encoding S. scabiei homologues of 3 house dust mite allergens-the M-177 apolipoprotein, glutathione S-transferase, and paramyosin--were identified. The availability of cDNA libraries from S. scabiei var. hominis and S. scabiei var. vulpes and the emerging public sequence databases from both opens up new possibilities in scabies research.

Multiple nuclear pseudogenes of mitochondrial DNA exist in the canine genome.

Many copies of nuclear counterparts of mitochondrial DNA (mtDNA) were found in nuclear DNA from sperm heads of the domestic dog, Canis familiaris, by DNA-DNA hybridization and DNA sequencing. Nuclear counterparts homologous to the mtDNA D-loop region were cloned into lambda phage vectors (EMBL4 and lambda gt11), and nucleotide sequences of seven different mtDNA pseudogenes were then determined. The seven pseudogenes were E3 (474 bp; 82% homology with canine mtDNA), E13 (1867 bp; 67%), 8B (2375 bp; 78%), 12A (2650 bp; 79%), 33 (4131 bp; 86%), 47 (4251 bp; 86%), and E17 (5721 bp; 71%). These seven mtDNA pseudogenes corresponded to portions of cytoplasmic mtDNA containing the genes ile, ND1, leu, 16S rRNA, val, 12S rRNA, phe, D-loop, pro, thr, cytb, and glu. A neighbor-joining phylogenetic tree constructed from 12S rRNA sequences in mtDNA pseudogenes 8B, 33, 47, and E17 and in 10 mtDNA fragments from other species showed that these four pseudogenes form a monophyletic clade with canine mtDNA. A neighbor-joining phylogenetic tree based on the 318-bp cytb region showed that the canine pseudogenes existed before the divergence of 17 related canids, and their divergence dates were calculated at around 4.4 to 8.6 million years ago.

Bacteriophage lambda and plasmid pUR288 transgenic fish models for detecting in vivo mutations.

We adapted transgenic rodent mutation assays based on fish carrying bacteriophage lambda and plasmid pUR288 vectors to address the needs for improved methods to assess health risks from exposure to environmental mutagens and also to establish new animal models to study in vivo mutagenesis. The approach entails separating the vectors from fish genomic DNA and then shuttling them into specialized strains of E. coli bacteria to analyze spontaneous and induced mutations in either lacI and cII or lacZ mutational targets. Fish exhibited low frequencies of spontaneous mutants comparable to the sensitivity of transgenic rodent models. Mutations detected after treating fish with chemical mutagens showed concentration-dependent, tissue-specific, and time-dependent relationships. Spontaneous and induced mutational spectra also were consistent with the specificity of known mutagens, further supporting the utility of transgenic fish for studies of in vivo mutagenesis.

Affinity Selection of DNA-Binding Proteins Displayed on Bacteriophage

Two transcription factors, human ATF1, its DNA-binding domain (ATF1BD), and the DNA-binding domain (GAL4BD) of the yeast GAL4 protein, were displayed on the surface of bacteriophage lambda vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, lambdafoo and lambdafooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4,000-fold over the vector phages. Further, the GAL4BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on lambdafooDc and lambdafoo, respectively. The ATF1BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on lambdafoo. These results suggest that the lambdafoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125.

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a lambda phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.

Background mutations and polymorphisms inlacZ-plasmid transgenic mice

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.

Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast.

We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.

Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination.

Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.