Modifying the mammalian genome by gene targeting

Over the past year, gene targeting in mammalian cells has become a facile technology. By using a variety of selection and screening protocols, it has become possible to direct modifications at the nucleotide level to specific genes, to target marker genes so that they become expressed under the control of endogenous promoters and to delete large regions of the genome.     

A series of vectors that simplify mammalian gene targeting

In order to facilitate the procedure of mammalian gene targeting, we have produced and functionally tested a series of generic vectors. Homologous recombination has been achieved with each vector. The vectors are designed for both replacement and insertional recombination, are suitable for hit and run strategies and contain all necessary genetic elements for both positive-negative and promoterless/gene fusion enrichment of homologous integrations. Multiple unique restriction sites are included to simplify the incorporation of genomic targeting sequences.     

Tissue-specific gene targeting by the multiprotein mammalian DREAM complex

The mammalian DP, RB-like, E2F, and MuvB-like proteins (DREAM) complex, whose key components include p130 and E2F4, plays a fundamental role in repression of cell cycle-specific genes during growth arrest. Mammalian DREAM is well conserved with Drosophila and Caenorhabditis elegans complexes that repress pivotal developmental genes, but the mammalian complex has been thought to exist only in quiescent cells and not to be linked with development. However, new findings here identify tissue-specific promoters repressed by DREAM in proliferating precursors, revealing a new connection between control of growth arrest and terminal differentiation. Mechanistically, tissue-specific promoter occupation by DREAM is dependent on the integrity of a repressor form of the SWI/SNF chromatin-remodeling complex.     

Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.     

Transient marker system for iterative gene targeting of a prototrophic fungus

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.     

Distorted segregation resulting from pea chromosome reconstructions with alien segments from Pisum fulvum

Pea (Pisum sativum L.) satellited chromosome reconstructions were analyzed by cytologic markers to identify segregation distortion events. The presence of modified chromosomes was evaluated on the basis of additional rDNA genes, an extra and a longer satellite, all derived from chromosome 5 and chromosome 7 from P. fulvum Sibth. & Sm. The segregation of modified satellited chromosome 5 was monitored through fluorescent in situ hybridization with rDNA probe; it fitted the expected 1:2:1 ratio after self-pollination of a heterozygous genotype for modified chromosome 5. In different genotypes, which were heterozygous for both modified chromosomes 5 and 7, the combined segregation of these chromosomes showed the occurrence of seven karyotype classes instead of the expected nine. The classes with modified chromosome 7 and without modified chromosome 5, whether heterozygous or homozygous, were absent. The hypothesis of gamete selection was rejected since the expected segregation ratio of 5:3:1 was significant by chi-square test. Based on the other hypothesis of postzygotic selection, the segregation ratio did not show a significant deviation from the expected 9:3:1 ratio, thereby indicating that embryo abortion caused the segregation distortion (SD). The hypothesis of the SD system involving two loci carried by the alien satellites of modified chromosomes 5 and 7 is discussed in relation to the evolution of the P. fulvum genome.     

Isoenzyme patterns of glutathione transferases from mammalian erythrocytes

The occurrence of glutathione transferase isoenzymes in mammalian erythrocytes was investigated. The enzymes present in the hemolysates of human, horse, beef, pig, and sheep erythrocytes were purified by a column of GSH-linked epoxy-activated Sepharose 6B and subjected to an isoelectric focusing run in the pH range 3.5-10. Human and horse preparations were resolved in a single peak of activity centered at pH 4.6 and 5.9, respectively. Two forms with a maximum of activity at pH 4.9 and 7.0 and four with a maximum at pH 5.9, 6.5, 7.1, and 8.1 were separated from bovine and porcine erythrocytes. At least six forms ranging from pH 4.3 to pH 7.1 were present in the ovine preparation, the neutral contributing more than 90% of total activity. The subunit composition of affinity-bound fractions was studied by sodium dodecyl sulfate-gel electrophoresis. The analysis revealed that erythrocyte glutathione transferases are composed of subunits of identical molecular weights. This result suggests that the polymorphism existing in beef, pig, and sheep may be due to charge isomers. The erythrocyte glutathione transferases did not express selenium-independent GSH peroxidase activity.     

Role of ooplasmic segregation in mammalian development

A new micromanipulation technique permitted the scrambling of the zygote cytoplasm. Such interference had no effect on preimplantation development, and when zygotes with scrambled cytoplasm were transfered to the pseudopregnant females, normal and fertile mice were born. This demonstrates that no morphogenetic factors are prelocalized in the egg cytoplasm. Cleavage characteristics of mouse embryos provide the evidence that zygote cytoplasm does not define any determinate type of cleavage. We conclude that the mechanism of ooplasmic segregation is not used in the mouse (and presumably mammalian) development. It is suggested that the turning point in the evolution of mammalian embryogenesis was the transition to the intrauterine development, that started the process leading among other changes, to the loss of the ooplasmic morphogenetic determinants.Correspondence to: S.V Evsikov     

Self-fertile apple resulting from S-RNase gene silencing

Self-incompatibility (SI) restricts fertilisation and fruit setting in many tree fruit crops. In apple, we have produced transgenic trees harbouring extra copies of the endogenous S-gene controlling SI. Two independent transgenic genotypes were characterised in detail. Controlled self- and cross-pollination of the flowers of trees from both genotypes over a 3-year-period showed that the transgenic lines produced normal levels of fruit and seeds after selfing. In contrast, the controls produced much less fruit following self- compared to cross-pollination. Fruit set data correlated with the results of microscopic evaluation of pollen tube growth through the pistil, which revealed inhibition after selfing in the controls but not in the transgenic lines. The self-fertile phenotype was associated with the complete absence of pistil S-RNase proteins, which are the products of the targeted S-gene. These results confirm that self-fertility was due to inhibition of expression of the S-RNase gene in the pistil, resulting in un-arrested self-pollen tube growth, and fertilisation.Communicated by P. Debergh     

The molecular basis of multiple vector insertion by gene targeting in mammalian cells

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.     

Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans.     

Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G₂) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant'' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death.     

Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA damaging agents

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway.     

Analysis of biological selections for high-efficiency gene targeting.

A two-marker selection system that allows the efficient isolation of diploid gene knockouts by two sequential rounds of targeted homologous recombination has been developed. A systematic evaluation of the biological parameters that govern the selection process showed that a successful strategy must match the expression level of the target gene, the efficacy of the marker, and the selection stringency. An enrichment ratio of 5,000- to 10,000-fold, which resulted in a 30% targeting efficiency of the c-myc gene in a fibroblast cell line, has been achieved. Such efficiency brings the difficulty of gene targeting effectively down to the level of simple transfections, since only 10 to 20 drug-resistant clones need to be screened to recover several homologous hits. The general utility of the targeting strategy is of interest to investigators studying gene function in a large variety of mammalian tissue culture systems.