Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

脂质体DC-Chol/DOPE对HEK293FT细胞的高效转染及其在腺病毒制备中的应用

重组病毒载体系统因为具有高效的基因转移能力得到了广泛应用,而病毒包装细胞的转染是重组病毒制备过程中的关键步骤。优化了脂质体DC-Chol/DOPE介导的转染常用的病毒包装细胞系HEK293FT的实验条件,比较了DC-Chol/DOPE、Lipofectamine2000和磷酸钙共沉淀法转染细胞的效率,并且比较了用DC-Chol/DOPE和磷酸钙共沉淀法转染293FT细胞制备重组腺病毒的结果,发现DC-Chol/DOPE对293FT细胞的转染效率以及最终收获的病毒滴度都远高于磷酸钙共沉淀法转染。所以,利用DC-Chol/DOPE转染293FT细胞制备重组病毒是一种简单、高效、成本低廉的方法。     

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.     

Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system

In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system. The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors [Singh et al., Gene 20 (1982) 441-449]. The model system studied was the TRP1 gene of S. cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene. ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast. Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast. The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively. The latter approaches the theoretical limit of 50%. In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold. This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation. For yeast genes, direct phenotypic selection is possible in the recipient strain.     

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.     

Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.     

Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells.

Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.     

Stability and expression of bacterial genes in replicating geminivirus vectors in plants.

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.     

Poly-L-ornithine-mediated transformation of mammalian cells.

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.     

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.     

A guide to choosing vectors for transformation of the plastid genome of higher plants

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3'-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3'-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

High-frequency transfection of mouse FM3A mammary carcinoma cells in suspension culture with plasmid DNA

High-frequency transfection of mouse FM3A cells, grown in suspension, with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 h followed by an osmotic shock with a hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this concentration range, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible, and carrier DNA was not required. The efficiency was about 100 times higher than that of the method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected.     

A new transfection method of mouse FM3A mammary carcinoma cells with plasmid DNA

High-frequency transfection of mouse FM3A cells with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 hours followed by an osmotic shock with hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this range of NaCl concentration, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible and carrier DNA was not required. The efficiency was about 100 times higher than that of the widely used method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected with different restriction sites.     

Variables Controlling the Expression Level of Exogenous Genes in Dictyostelium

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.     

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.     

Variables controlling the expression level of exogenous genes in Dictyostelium.

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.     

Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes.

We sought an efficient means to introduce specific human chromosomes into stable interspecific hybrid cells for applications in gene mapping and studies of gene regulation. A defective amphotropic retrovirus was used to insert the gene conferring G418 resistance (neo), a dominant selectable marker, into the chromosomes of diploid human fibroblasts, and the marked chromosomes were transferred to mouse recipient cells by microcell fusion. We recovered five microcell hybrid clones containing one or two intact human chromosomes which were identified by karyotype and marker analysis. Integration of the neo gene into a specific human chromosome in four hybrid clones was confirmed by segregation analysis or by in situ hybridization. We recovered four different human chromosomes into which the G418 resistance gene had integrated: human chromosomes 11, 14, 20, and 21. The high efficiency of retroviral vector transformation makes it possible to insert selectable markers into any mammalian chromosomes of interest.