Retro-recombination screening of a mouse embryonic stem cell genomic library

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for ‘rolling out’ plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage–plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed ‘retro-recombination screening’ (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a λTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage λ into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.     

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination

Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.     

Bacteriophage gene targeting vectors generated by transplacement

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.     

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.     

Modulation of EcoKI restriction in vivo: role of the lambda Gam protein and plasmid metabolism.

Two novel types of alleviation of DNA restriction by the EcoKI restriction endonuclease are described. The first type depends on the presence of the gam gene product (Gam protein) of bacteriophage lambda. The efficiency of plating of unmodified phage lambda is greatly increased when the restricting Escherichia coli K-12 host carries a gam+ plasmid. The effect is particularly striking in wild-type strains and, to a lesser extent, in the presence of sbcC and recA mutations. In all cases, Gam-dependent alleviation of restriction requires active recBCD genes of the host and recombination (red) genes of the infecting phage. The enhanced capacity of Gam-expressing cells to repair DNA strand breaks might account for this phenomenon. The second type is caused by the presence of a plasmid in a restricting host lacking RecBCD enzyme. Commonly used plasmids such as the cloning vector pACYC184 can produce such an effect in strains carrying recB single mutations or in recBC sbcBC strains. Plasmid-mediated restriction alleviation in recBC sbcBC strains is independent of the host RecF, RecJ, and RecA proteins and phage recombination functions. The presence of plasmids can also relieve restriction in recD strains. This effect depends, however, on the RecA function in the host. The molecular mechanism of the plasmid-mediated restriction alleviation remains unclear.     

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.     

Jekyll, a family of phage-plasmid shuttle vectors

A series of shuttle vectors has been constructed, which consist of a plasmid carrying a polylinker sequence and an M13 origin integrated into a lambda vector. A short direct repeat flanking the plasmid allows plasmid excision by homologous recombination. Sequences are cloned into unique restriction sites within the plasmid, and can be recovered either in phage or plasmid form, or can be packaged further as single-stranded DNA phage. These vectors therefore combine the efficiency of phage lambda cloning and screening with the ease of handling or analysing plasmid or M13 clones.     

Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neoR colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.     

Targeted modification of the complete chicken lysozyme gene by poxvirus-mediated recombination.

We have developed a novel ex vivo system for the rapid one-step targeted modification of large eucaryotic DNA sequences. The highly recombinant environment resulting from infection of rabbit cornea cells with the Shope fibroma virus was exploited to mediate precise modifications of the complete chicken lysozyme gene domain (21.5 kb). Homologous recombination was designed to occur between target DNA (containing the complete lysozyme gene domain) maintained in a lambda bacteriophage vector and modified targeting DNA maintained in a plasmid. The targeting plasmids were designed to transfer exogenous sequences (for example, beta-galactosidase alpha-complement, green fluorescent protein, and hydrophobic tail coding sequences) to specific sites within the lysozyme gene domain. Cotransfection of the target phage and a targeting plasmid into Shope fibroma virus infected cells resulted in the poxvirus-mediated transfer of the modified sequences from plasmid to phage. Phage DNA (recombinant and nonrecombinant) was then harvested from the total cellular DNA by packaging into lambda phage particles and correct recombinants were identified. Four different gene-targeting pairings were carried out, and from 3% to 11% of the recovered phages were recombinant. Using this poxvirus-mediated targeting system, four different regions of the chicken lysozyme gene domain have been modified precisely by our research group overall with a variety of inserts (6-971 bp), deletions (584-3000 bp), and replacements. We have never failed to obtain the desired recombinant. Poxvirus-mediated recombination thus constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise modification of large eucaryotic gene domains when compared with traditional practices.     

Transactivation of p'R promoter of phage lambda

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

New plasmid vectors for specific gene targeting in Spiroplasma citri

In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative, oriC plasmid pBOT1 as the disruption vector. However, plasmid recombination required extensive passaging of the transformants and, in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore, to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described.     

Structure of a recombination site in the transducing bacteriophage lambda plac5 DNA

A recombination site in the transducing bacteriophage lambda plac5 DNA has been structurally elucidated. Comparison of primary structures of E. coli lac-operon (distal end of lacZ gene, Z-Y spacer, and proximal end of lacY gene) described earlier with corresponding segments of bacteriophages lambda CI857 and lambda plac 5-2 DNAs sequenced in this paper showed that the bacterial DNA insert ends immediately after Z-Y spacer, just before the initiating triplet ATG of lacY gene. It thus follows that in contrast to the earlier conception, the insert does not seem to include any part of lacY gene. The recombination sites in both phage and bacterial DNA contain structurally homological segments about 20 b. p. long (crossover region), with two extra basepairs in the bacterial DNA (AT in the sense-strand). We suppose that the very dinucleotide plays a substantial role in initiation of recombinational event: causing formation of a nonperfect heteroduplex structure, it determines the T-A internucleotide bond to be endonucleolytically cut (crossover point) followed by exonucleolytic elimination of the extra links (AT) and reciprocal strand exchange. The second recombination site in lambda plac5 DNA has been localized by us within lacI gene as being close to the HindII site (nucleotides 854 to 859 of the gene). The structures of the two regions of site-specific recombination may shed light upon mechanisms of the phage abnormal excision leading to formation of transducing phages.     

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.     

Gene fusion in vivo using transductional cointegrates

A method is described which allows the efficient construction of hybrids between homologous genes. The technique is based on the phenomenon of cointegrate transduction in which the homology between cloned sequences present on a bacteriophage lambda vector and a plasmid vector is exploited to allow the packaging of a plasmid-phage recombinant. The size of the cointegrate molecule can be far beyond the normal packaging limit of lambda and still allow the transduction of plasmid-borne drug-resistance markers. This method allows the exchange of the 5' and 3' ends of the participating genes as well as the exchange of sequences residing between the end-points of homology between the two genes. Hybrids of either type were constructed between a sea urchin and a Drosophila actin gene using the transductional cointegrate method in vivo. This approach does not require the use of specialized phage or plasmid vectors and can also be used to screen plasmid libraries with a bacteriophage lambda probe.     

A role for recombination in the production of "free-loader" lambda bacteriophage particles

Density-labeled crosses were performed with bacteriophage lambda under conditions which diminish DNA duplication. The production of viable phage containing fully conserved parental DNA was found to be dependent upon the action of the genetic recombination systems. The production of phage containing DNA with one newly synthesized chain was less dependent upon recombination. The production of phage with chromosomes both of whose chains were synthesized following infection show little, if any, dependence on recombination. One can speculate that some step in the maturation process of bacteriophage lambda is inseparable from the reduction of lambda DNA to the monomeric rods characteristic of lambda virions.     

Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate – strand annealing or strand invasion – accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.     

Mutational specificity studies of endogenous mammalian cell loci: methodological aspects

We report here the details of a modified cloning method first described by Seed et al. (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells. The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage lambda vector. Recombinant phage form blue plaques on a non-suppressor, lacZ amber host. We have determined the sensitivity of the method. Homologous recombination frequency is in the order of 10(-3), 6-7 orders of magnitude above non-homologous events. Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector. A background of plaques is observed along with the desired blue plaques. Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events.     

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.