Replacement Vector Research Articles

Requirements for incorporation of Pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles

The roles of the human immunodeficiency virus precursor polyproteins Pr55gag and Pr160gag-pol in viral core assembly were studied in CMT3-COS cells. To do this, the precursors were expressed separately by using a simian virus 40 late replacement vector system described previously. Consistent with previously published data, our results show that the Pr55gag precursor, when expressed alone, was able to form particles which had an immature morphology and that particle formation required the presence of a myristate addition signal at the amino terminus of the precursor. In contrast, the Pr160gag-pol precursor was not able to form particles when expressed alone, although it still underwent proteolytic processing. Coexpression of the two precursor polyproteins from separate vectors in the same cell resulted in processing of the Pr55gag in trans by the protease embedded in Pr160gag-pol and the formation of virus-like particles containing the products of both precursors. Proteolytic processing occurred independently of the presence of a functional myristate addition signal on either precursor. On the other hand, removal of myristate from one or the other precursor had nonreciprocal effects on virus particle formation. Cells expressing Pr55gag lacking myristate and Pr160gag-pol containing it did not produce particles. Cells expressing a myristylated Pr55gag and unmyristylated Pr160gag-pol still produced virus-like particles which contained nearly normal amounts of Pr160gag-pol. The results suggest that the incorporation of Pr160gag-pol into particles is largely determined by intermolecular protein-protein interactions between the two precursor polypeptides.

Construction of gene replacement vectors for Gram? bacteria using a genetically modified sacRB gene as a positve selection marker

Based on the wide-host-range suicide vector pSUP102 (Simon et al. 1983), gene replacement vectors (pWS232/pWS233) have been developed that facilitate the identification by a positive selection procedure of double recombination events in Gram− bacteria. The vectors contain the tetracycline and gentamicin resistance gene as selectable markers, as well as a modified sacRB gene mediating sucrose sensitivity. In order to increase the versatility of the sacRB gene as a positive selection marker and, hence, of vectors that carry this gene, an EcoRI as well as a HindIII site located within the coding region of the sacRB gene were removed by in-vitro mutagenesis. To test the suitability of the vectors, a Tn5-carrying EcoRI fragment of Rhizobium leguminosarum biovar. viciae VF39 was homogenotized into the wild-type strain, resulting in double recombinants with a Lac− phenotype. Although the Tn5 insertion was flanked on one side by only approximately 100 bp of VF39 homologous DNA, this was sufficient for homologous recombination to occur, and double recombinants could readily be isolated.

Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors.

Five replacement vectors (RV) and one insertion vector (IV) were constructed in which ca. 10 kb of genomic Gi2 alpha sequence, flanked on one (IV) or both (RV) sides by a thymidine kinase (TK) marker, were disrupted by a Neo marker inserted into the NcoI site of exon 3. G418RFIAUR clones corresponding to ca. 4 x 10(8) ES cells electroporated with replacement vectors were analyzed and revealed no targeting event. The insertion vector, however, was integrated by a single reciprocal recombination resulting in a duplication of homology (Hit step; G418RFIAUS), which was lost--together with the plasmid and the TK sequences--by intrachromosomal recombination (Run step; G418RFIAUR). Thus, the Hit and Run strategy can be used with a selectable marker disrupting the targeted gene, giving rise to the same targeted product that would have been expected to arise from a double crossover with a replacement vector.

A new λ RES vector with a built-in Tn1721-encoded excision system

A new λ replacement vector for construction of genomic libraries was developed which allows the excision of cloned fragments by site-specific recombination from the λ DNA and conversion into autonomously replicating plasmids. The vector system, derived from λEMB L4, is called λRES. It contains two recognition sites for site-specific recombination from Tn1721 on both sides of the replacement fragment of λEMBL4. Additionally, on one side, there is a plasmid replication origin from Rtsl with a kanamycin-resistance (Km R) marker. DNA fragments in the range of 8–14kb may be inserted between BamHI or SalI sites in the λ vector. Efficient excision and conversion of plaque-forming units into Km R colonies are obtained by infection of Escherichia coli strains harbouring Tl739 tnpR on a F′ plasmid. Tn l739tnpR is a derivative of Tn1721 with a chloramphenicol-resistance-encoding gene (Cm R), the λ cl repressor gene, and a further copy of the resolvase-encoding tnpR gene under control of the tac promoter.

Bacteriophage lambda as a cloning vector.

Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

Local-geometric-projection method for noise reduction in chaotic maps and flows.

We describe a method for noise reduction in chaotic systems that is based on projection of the set of points comprising an embedded noisy orbit in ${\mathit{openR}}^{\mathit{d}}$ toward a finite patchwork of best-fit local approximations to an m-dimensional surface M'\ensuremath{\subset}${\mathit{openR}}^{\mathit{d}}$, m\ensuremath{\le}d. We generate the orbits by the delay coordinate construction of Ruelle and Takens [N. H. Packard et al., Phys. Rev. Lett. 45, 712 (1980); F. Takens, in Dynamical Systems and Turbulence, Warwick, 1980, edited by D. A. Rand and L.-S. Young (Springer, Berlin, 1981)] from time series v(t), which in an experimental situation we would assume to have come, together with additional high-dimensional background noise, from an underlying dynamical system ${\mathit{f}}^{\mathit{t}}$: M\ensuremath{\rightarrow}M existing on some low m-dimensional manifold M. The surface M' in ${\mathit{openR}}^{\mathit{d}}$ is the assumed embedded image of M. We give results of systematic studies of linear (tangent plane) projection schemes. We describe in detail the basic algorithm for implementing these schemes. We apply the algorithm iteratively to known map and flow time series to which white noise has been added. In controlled studies, we measure the signal-to-noise ratio improvements, iterating ${\mathit{n}}_{\mathit{M}}$ times until a stable maximum ${\mathrm{\ensuremath{\delta}}}_{\mathit{M}}$ is achieved. We present extensive results for ${\mathrm{\ensuremath{\delta}}}_{\mathit{M}}$ and ${\mathit{n}}_{\mathit{M}}$ for a wide range of values of embedding trial dimension d, projection dimension k, number of nearest-neighbor points for local approximation \ensuremath{\nu}, embedding delay \ensuremath{\Delta}, sampling interval \ensuremath{\Delta}T, initial noise amplitude scrN, and trajectory length N.We give results for very low and very high noise amplitudes 0%\ensuremath{\le}scrN\ensuremath{\le}100%. We develop an empirical method for estimating the initial noise level for a given experimental time series, and for the optimal choice of algorithm parameters to achieve peak reduction. We present interesting results of application of the noise-reduction algorithm to a chaotic time series produced from a periodically driven magnetoelastic ribbon experiment on the control of chaos. Two noteworthy elements of the noise-reduction method we describe result in certain stabilizing and efficiency features. The first is our use of a physical replacement time series, which is a unique scalar time series with the property that its corresponding delay coordinate construction data state vector time series in ${\mathit{openR}}^{\mathit{d}}$ is optimally close to the noise-reduced replacement vector time series generated by the projection. The second is the introduction of a ``measure-ordered'' cover, which produces notable improvement in reliability, control, and computational efficiency of the whole algorithm.

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25 S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.

Mutations were targeted to the Hprt locus of mouse embryo-derived stem cells by using 22 different sequence replacement and sequence insertion vectors. The targeting frequency was examined at two sites within the Hprt locus as a function of the extent of homology between the targeting vector and the target locus. The targeting frequency was also compared by using vectors prepared from isogenic and nonisogenic DNA sources. With one exception, all of the vectors showed the same exponential dependence of targeting efficiency on the extent of homology between the targeting vector and the target locus. This was true regardless of whether they were sequence replacement or sequence insertion vectors, whether they were directed toward either of the two different sites within the Hprt locus, or whether they were prepared from isogenic or nonisogenic DNA sources. Vectors prepared from isogenic DNA targeted four to five times more efficiently than did the corresponding vectors prepared from nonisogenic DNA. The single case of unexpectedly low targeting efficiency involved one of the vectors prepared from nonisogenic DNA and could be attributed to an unfavorable distribution of heterology between the Hprt sequences present in the targeting vector and the endogenous Hprt gene.

Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.

Mutations were targeted to the Hprt locus of mouse embryo-derived stem cells by using 22 different sequence replacement and sequence insertion vectors. The targeting frequency was examined at two sites within the Hprt locus as a function of the extent of homology between the targeting vector and the target locus. The targeting frequency was also compared by using vectors prepared from isogenic and nonisogenic DNA sources. With one exception, all of the vectors showed the same exponential dependence of targeting efficiency on the extent of homology between the targeting vector and the target locus. This was true regardless of whether they were sequence replacement or sequence insertion vectors, whether they were directed toward either of the two different sites within the Hprt locus, or whether they were prepared from isogenic or nonisogenic DNA sources. Vectors prepared from isogenic DNA targeted four to five times more efficiently than did the corresponding vectors prepared from nonisogenic DNA. The single case of unexpectedly low targeting efficiency involved one of the vectors prepared from nonisogenic DNA and could be attributed to an unfavorable distribution of heterology between the Hprt sequences present in the targeting vector and the endogenous Hprt gene.

High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors.

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.

High-Fidelity Gene Targeting in Embryonic Stem Cells by Using Sequence Replacement Vectors

Mutations were targeted to the Hprt locus in murine embryonic stem cells by using sequence replacement vectors. When the vector was designed such that the mutated sequences were flanked on both sides by several kilobases of DNA homologous to the target locus, replacement of chromosomal sequences with the exogenous DNA occurred with precision. If, on the other hand, the target-homologous DNA on one arm of the vector was reduced to below 1 kb in length, the fidelity of recombination was diminished.

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.

The construction of recA–deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk–assessment studies

AbstractA vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA‐). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.

Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.

Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors.

Replacement vectors with genomic DNA originating from different mouse strains were used to introduce site-specific mutations into the creatine kinase M (CKM) gene of mouse embryonic stem (ES) cells. Here we demonstrate that in mouse strain 129-derived ES cells, the gene is at least 25-fold more efficiently targeted with an isogenic, 129-derived vector (129-pRV8.3) than with a nonisogenic, BALB/c-specific vector (BALB/c-pRV8.3). The two targeting constructs were identical except for allelic differences which were typed by partial sequencing. These included base pair mismatches (2%) and a polymorphic [GTC]-repeat length variation. Both in separate transfections as well as in cotransfections with mixed vectors, homologous disruption of the CKM gene resulted uniquely from the 129-isogenic DNA. Our data confirm earlier observations on requirements for homologous recombination in pro- and eukaryotic systems and indicate that targeting of the CKM locus is highly sensitive to small sequence differences between cognate segments in the endogenous and incoming DNA.

Bacteriophage lambda as a cloning vector.

Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

The length of homology required for gene targeting in embryonic stem cells.

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.

The length of homology required for gene targeting in embryonic stem cells.

Homologous recombination has been used to introduce site-specific mutations into murine embryonic stem (ES) cells with both insertion and replacement vectors. In this study, we compared the frequency of gene targeting with various lengths of homology and found a dramatic increase in targeting with an increase in homology from 1.3 to 6.8 kb. We examined in detail the relationship between the length of homology and the gene-targeting frequency for replacement vectors and found that a critical length of homology is needed for targeting. Adding greater lengths of homology to this critical length has less of an effect on the targeting frequency. We also analyzed the lengths of homology necessary on both arms of the vector for gene replacement events and found that 472 bp of homology is used as efficiently as 1.2 kb in the formation and resolution of crossover junctions.

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.