Genome Of Mammalian Cells Research Articles

Molecular Analysis of Chromosomal Rearrangements in Mammalian Cells After øC31-Mediated Integration

Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.

Purification of retroviral vectors for clinical application: Biological implications and technological challenges

For centuries mankind led a difficult battle against viruses, the smallest infectious agents at the surface of the earth. Nowadays it is possible to use viruses for our benefit, both at a prophylactic level in the production of vaccines and at a therapeutic level in the promising field of gene therapy. Retroviruses were discovered at the end of the 19th century and constitute one of the most effective entities for gene transfer and insertion into the genome of mammalian cells. This attractive feature has intensified research in retroviral vectors development and production over the past years, mainly due to the expectations raised by the concept of gene therapy. The demand for high quality retroviral vectors that meet standard requisites from the regulatory agencies (FDA and EMEA) is therefore increasing, as the technology has moved into clinical trials. The development of safer producer cell lines that can be used in large-scale production will result in the production of large quantities of retroviral stocks. Cost-efficient and scalable purification processes are essential for production of injectable-grade preparations to achieve final implementation of these vectors as therapeutics. Several preparative purification steps already established for proteins can certainly be applied to retroviral vectors, in particular membrane filtration and chromatographic methods. Nevertheless, the special properties of these complex products require technological improvement of the existing purification steps and/or development of particular purification steps to increase productivity and throughput, while maintaining biological activity of the final product. This review focuses on downstream process development in relation to the retroviral vectors characteristics and quality assessment of retroviral stocks for intended use in gene therapy.

792. Post-Integrative Gene Silencing in the Sleeping Beauty Transposition System

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is one of the most efficient non-viral integrating systems described to date, all previous estimations of its integrative potential have relied on the use of antibiotic selection schemes. Based on previous studies demonstrating post-integrative gene silencing with other gene transfer systems (e.g. retroviruses), we investigated how genomic integration influenced transposon expression in human cells.

388. Chromosomal Rearrangements in Mammalian Cells Are Induced at High Frequency by the Bacteriophage Derived Integrase phiC31

Top of pageAbstract Recent reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that the integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosomes. However, there is strong evidence that cryptic lox sites for Cre mediated integration are present in the eukaryotic genome which result in chromosomal translocations. Thus, we investigated whether or not cryptic phiC31 recognition sites may result in chromosomal rearrangements. In this study we found that the integrase phiC31 induced chromosomal rearrangements in the mammalian host genome with a frequency of up to 13% as demonstrated by integration site analysis and limited karyotyping studies. Single cell clones from in human hepatoma cells (Huh-7), human embryonic kidney cells (293), and the human colon carcinoma cell line HCT116 were grown and sites of insertion were characterized based on an established plasmid rescue protocol which allows identification of both chromosomal DNA sequences flanking the integrated transgene. Out of 81 independent integration events analyzed, 7 (8.6%) integrated into chromosome 19 (19q13.31) and 9 (11%) into chromosome 12 (12q22). Most importantly, of all independent integration sites analyzed 13% were found to contain an integrated transgene which was flanked by DNA sequences originated from two different chromosomes. This observation strongly suggested that chromosomal rearrangements might have occurred. This assumption is further supported by the fact that if one uses the same experimental approach this phenomenon has not been reported in over 1000 Sleeping Beauty mediated integration events and in less than 0.25% (1 out of 400) of all sites of insertion after adeno-associated virus (AAV) mediated integration. To confirm chromosomal translocations, we determined the genotype of single cell clones by karyotype analysis in HCT116 cells, a cell line for which the chromosomal content is relatively stable and well characterized. In sharp contrast to wildtype HCT116 cells, all twenty-four metaphases examined in cell clone HCT-1A7 had the same chromosomal abnormality on chromosome 22 band q13, which confirmed our plasmid rescue data. For some clones, a mixture of transgenes flanked by DNA sequences originated from the same but also two different chromosomes was identified. Taken together, his feature will introduce novel ways to genetically modify mammalian cells and to study the fate of cells with chromosomal translocations but suggests that additional safety studies will be required before the use of this integrase in gene therapy approaches.

Can the state of cancer chemotherapy resistance be reverted by epigenetic therapy?

BackgroundTranscriptome analysis shows that the chemotherapy innate resistance state of tumors is characterized by: poorly dividing tumor cells; an increased DNA repair; an increased drug efflux potential by ABC-transporters; and a dysfunctional ECM. Because chemotherapy resistance involves multiple genes, epigenetic-mediated changes could be the main force responsible of this phenotype. Our hypothesis deals with the potential role of epigenetic therapy for affecting the chemotherapy resistant phenotype of malignant tumors.Presentation of the hypothesisRecent studies reveal the involvement of DNA methylation and histone modifications in the reprogramming of the genome of mammalian cells in cancer. In this sense, it can be hypothesized that epigenetic reprogramming can participate in the establishment of an epigenetic mark associated with the chemotherapy resistant phenotype. If this were correct, then it could be expected that agents targeting DNA methylation and histone deacetylation would by reverting the epigenetic mark induce a global expression profile that mirror the observed in untreated resistant cells.Testing the hypothesisIt is proposed to perform a detailed analysis using all the available databases where the gene expression of primary tumors was analyzed and data correlated with the therapeutic outcome to determine whether a transcriptome profiling of "resistance" is observed. Assuming an epigenetic programming determines at some level the intrinsic resistant phenotype, then a similar pattern of gene expression dictated by an epigenetic mark should also be found in cell acquiring drug resistance. If these expectations are meet, then it should be further investigated at the genomic level whether these phenotypes are associated to certain patterns of DNA methylation and chromatin modification. Once confirmed the existence of an epigenetic mark associated to either the intrinsic or acquired chemotherapy resistant phenotype, then a causal association should be investigated. These preclinical findings should also be tested in a clinical setting.Implications of the hypothesisOur hypothesis on the ability of epigenetic therapy to revert the epigenetic changes leading to a transcritome profile that defines the resistant state will eventually be a more rational and effective way to treat malignant tumors.

573. Investigation into the Frequency of Post-Integrative Gene Silencing Using a Non-Selective Sleeping Beauty Transposition System

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single plasmid (pT/RSV-YFP.CMV-SB) containing both a YFP-marked transposon and a separate transposase expression cassette (external to the transposon), then transfected the plasmid into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate approximately 100 clonal cell lines. After 8 weeks of growth (>50 cell divisions) 30% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy and FACS analyses. We have screened these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Results indicate approximately 50% of the total cell lines contain an integrated transposon sequence(s), of which roughly 20% show little or no YFP expression. Our early results, based upon Hpa II (a methylation sensitive restriction enzyme) cleavage of genomic DNA, suggested the lack of YFP expression was not due to DNA methylation but to some other regulatory system. However, more recent data (including the use of the methylation specific McrBC enzyme) indicates that some of the integrated transposons do in fact become methylated over time. Possibly related, most individual cell lines show heterogeneous YFP levels throughout the cell population. Using aphidicolin (a cell cycle inhibitor) we determined that the varied YFP expression is not cell cycle dependent. We are continuing to explore post-integrative gene expression/silencing using a variety of approaches including a detailed analysis of how CpG concentration within the transposon expression cassette affects long-term gene expression. Overall, our transposon clones are proving to be an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of transcriptional silencing of integrating gene transfer vectors.

1056. Suppression and Enhancement of Gene Repair by the Modulation of ATM Function in Mammalian Cells

Targeted gene repair utilizes oligonucleotides to direct single base alterations in the genomes of mammalian cells. These vectors are designed to hybridize to the site of interest and to create a single mismatch with the mutant base. Although the mechanism of the nucleotide exchange phase of the reaction remains to be fully elucidated, evidence suggests that the induction of homologous recombination (HR) in response to DNA damage elevates DNA pairing activity and results in increased levels of gene repair. The HR pathway is controlled by the activation of the cell cycle checkpoint regulator, Ataxia-Telangiectasia mutated (ATM), which is induced by either DNA damage or by the presence of single stranded DNA. The radio-sensitizer, caffeine, has previously been shown to inhibit ATM kinase activity, by interfering with the phosphorylation of downstream substrates. When caffeine was added to a gene repair reaction prior to the introduction of the oligonucleotide, the frequency of correction was reduced, reinforcing the importance of HR in gene repair. If, however, caffeine was added after the oligonucleotide, the correction frequency was enhanced suggesting a dual functionality for caffeine in the gene repair reaction. Thus, we utilize caffeine as a reagent and a tool to understand the regulation of this reaction in greater detail. ATM activation in cells exposed to 4mM caffeine was examined following oligonucleotide delivery. At 18 and 24 hours, the level of ATM activation was increased significantly and even at 48 hours post-delivery, when ATM activation form the introduction of the ssDNA vector disappears, activated ATM was still quite discernable in caffeine treated cells. This sustained ATM activation could be explained by a concurrent effect of caffeine on the gene repair reaction; the oligonucleotide is retained by the cell for a longer period than in untreated cells. At 16 hours post electroporation, both cell populations exhibited about a 70% uptake of the oligonucleotide (as measured by the fluorescence of a FAM conjugated oligonucleotide). However, between 16 and 48 hours, the non-treated cells exhibit a steep drop in oligonucleotide levels, as compared to the caffeine treated cells. This suggests that the oligonucleotide activates ATM in all cells but in cells treated with caffeine, it reactivates ATM through an extended cellular half-life. The use of caffeine as a tool for studying the regulatory pathways of gene repair lead us to conclude that the activation of ATM is a critical step in promoting successful gene correction. On one hand ATM is required to induce HR which catalyses the first phase of gene repair (DNA pairing), but on the other hand, activated ATM can block the replication of corrected cells affecting the overall frequency of the reaction. We are now modulating the levels of ATM so that the balance can be tipped in favor of more robust levels of gene repair.

Montagna symposium on epidermal stem cells oligonucleotide-directed gene correction in epidermis.

Oligonucleotide-directed gene alteration produces a targeted DNA sequence change in the genome of mammalian cells. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. Reliable, sensitive, and standardized assays played a critical role in the measurement of gene correction frequency among different cell types and in evaluating the structure-activity relationship of oligonucleotides. Mechanistic studies using these assays have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The epidermis is an ideal tissue where oligonucleotides can be administered locally and the treated sites can be monitored easily. But given the low frequency of gene correction, general selection procedures and amplification of corrected cells via epidermal stem cells are ultimately needed to make the gene repair technology practical. Recent data suggest that the in vivo application of oligonucleotides may be capable of gene correction in epidermal stem cells and the subsequent expansion of the corrected cells may result in an apparent high-level and long-lasting gene repair. Advances in oligonucleotide delivery and targeting of epidermal stem cells will be required for potential application of oligonucleotides toward treatment of genodermatoses.

816. Development of Potent DNA Vaccines by Coupling of Linear MIDGE Vectors to Peptides

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single autonomously integrating plasmid (pT/YFP-SB) containing a YFP-marked transposon and an external plasmid-encoded transposase expression cassette, and transfected it into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate a total of 183 clonal cell populations. Unexpectedly, after 6 weeks of growth (> 30 cell divisions) 28% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy. We have begun screening these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Interestingly, preliminary results indicate that up to 15% of the total cell lines contain non-expressing integrated transgenes and we are currently investigating whether this may be due to post-integrative gene silencing. Surprisingly, up to 40% of the expressing cell lines contain persisting (non-dividing) episomal plasmid, as determined by DpnI/Southern blot analyses. Consequently, we are investigating whether co-delivery of SB and transposon sequences can facilitate episomal persistence in mammalian cells, possibly through transposase-mediated nuclear retention. Also of note, many individual cell lines showed heterogeneous YFP levels throughout the cell population. Currently, we are testing whether expression from an integrated transposon varies with the cell cycle, as has been seen in other integrating systems. The generation of stably silenced transposon clones provides an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of Sleeping Beauty biology.

811. The Hotspots of Integration Targeted by the Site-Specific Integrase PhiC31 May Be Cell Line Dependent

Recent reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that the phage integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosome. However, a study which carefully analyzes the consequences of phage integrase mediated integration for each independent integration event has yet to be performed. In the present study we characterized the chromosomal integration sites after phiC31 mediated integration from extrachromosomal plasmids in the two liver derived cell lines Huh7 (human hepatoma cell line) and Hep1A (mouse hepatoma cell line) and the human embryonic kidney cell line 293. The number of cell clones selected in a colony forming assay from all three cell lines revealed a 6.0 fold, 17.2 fold and 4.5 fold increase in the number of colonies in Huh7 cells, Hep1A cells and 293 cells compared to the mutant integrase control group, respectively. Single cell clones from all three cell lines were amplified and the insertion sites were identified and characterized based on a plasmid rescue protocol. We found frequent smaller deletions of up to 26 bp in the chromosomal target sites (13 of 32 integrations) and deletions of up to 10 bp in the phage integrase attachment site attB (8 of 32 integrations) in both, Huh-7 and 293 cells. Surprisingly, we also detected insertions of up to 177 bp (7 of 32 integrants); most of the insertions were < 4 bp. The origin of these inserted DNA sequences remains to be determined. Out of the 32 independent integration events analyzed so far, three integrated into human chromosome 19 (19q13.31), a site not previously reported. Because these initial results do not yet have a statistically significant number of independent observations, further analyses of additional clones from all three cell lines are ongoing. In summary, these results suggest that hotspots in the host genome targeted by the site-specific integrase phiC31 may depend on the cell line and perhaps on the tissue from which the cell line is derived. These findings will have important implications on improving the safety of ex vivo and in vivo gene therapy approaches in which stable and long-term transgene expression levels are required.

Регуляция изменчивости генома соматических клеток млекопитающих под влиянием экзогенных биологических факторов

Регуляция изменчивости генома соматических клеток млекопитающих под влиянием экзогенных биологических факторов

Non-homologous DNA end joining.

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.

Non-homologous DNA end joining.

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.

Ribozyme-mediated revision of RNA and DNA.

The splicing activity of Group I and Group II introns can be harnessed as a molecular tool that may potentially revise any gene of interest. Although hurdles remain, progress has been made in the development of these ribozymes for therapeutic application. While gene therapy is showing promise in the treatment of some diseases, many genes are regulated in ways that are not easily recapitulated by adding back a functional copy of the gene. Therefore, as an alternative to the traditional approach, it may be possible to correct the genetic information directly in the genome or in the messages transcribed from mutant genes. In this way, genetic information is left intact so that the cell may properly regulate the expression and processing of genetic information. Several classes of catalytic RNAs, or ribozymes, have the capacity to revise genetic information. Group I and Group II ribozymes are derived from naturally occurring Group I and Group II introns, respectively (1). These introns are found in the genes of a variety of lower eukaryotes and prokaryotes. They differ fundamentally from spliceosomal introns since Group I and Group II introns self-splice from the precursor RNA independent of the spliceosome. The intron adopts the catalytic structure that is capable of cleaving RNA splice sites and ligating the flanking exons together. In addition to self-splicing from RNA precursors, some Group II introns are able to reverse-splice into DNA. The splicing activity of these introns can be harnessed as a molecular tool that may potentially revise any gene of interest. The therapeutic revision of RNA by ribozymes is mechanistically different from that of DNA. To revise RNA sequences, a Group I intron from Tetrahymena thermophila has been adapted for trans-splicing (2). Trans-splicing ribozymes splice therapeutic RNA sequences onto a target transcript in a process called ribozyme-mediated RNA repair. Considerable progress has been made in the development of trans-splicing ribozymes for therapeutic applications such as treating sickle cell disease and cancer. The spliceosome has also been shown to trans-splice RNA transcripts (3). Spliceosomal-mediated RNA trans-splicing (SMaRT) is discussed by Mariano Garcia-Blanco (4) in this Perspective series. To modify genomic DNA, a Group II ribozyme has recently been developed that reverse-splices into target genes of interest (5, 6). If applicable to mammalian cell genomes, this exciting technology may allow the therapeutic insertion of genetic information into a specific target locus. In this Perspective series article, we report on the progress made in development of Group I and Group II ribozymes as tools to revise RNA and DNA. We will also discuss the hurdles that must be overcome to make clinical use of these molecular therapeutics.

Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells.

Mitochondrial fusion may regulate mitochondrial morphogenesis and underlie complementation between mitochondrial genomes in mammalian cells. The nuclear encoded mitochondrial proteins Mfn1 and Mfn2 are human homologues of the only known protein mediators of mitochondrial fusion, the Drosophila Fzo GTPase and Saccharomyces cerevisiae yFzo1p. Although the Mfn1 and Mfn2 genes were broadly expressed, the two genes showed different levels of mRNA expression in different tissues. Two Mfn1 transcripts were detected at similar levels in a variety of human tissues and were dramatically elevated in heart, while Mfn2 mRNA was abundantly expressed in heart and muscle tissue but present only at low levels in many other tissues. Human Mfn1 protein localized to mitochondria and participated in a high molecular weight, detergent extractable protein complex. Forced expression of Mfn1 in cultured cells caused formation of characteristic networks of mitochondria. Introduction of a point mutation in the conserved G1 region of the predicted GTPase domain (Mfn1K88T) dramatically decreased formation of mitochondrial networks upon Mfn1 overexpression, suggesting that network formation required completion of the Mfn1 GTPase cycle. Conversely, a protein variant carrying a point mutation in the G2 motif of the Mfn1 GTPase domain acted as a dominant negative: overexpression of Mfn1T109A resulted in fragmentation of mitochondria. We propose that Mfn1T109A interferes with fusion activity of endogenous Mfn1 protein, possibly by binding necessary cofactors, so to allow unopposed mitochondrial fission. Thus, Mfn1 appears to be a key player in mediating mitochondrial fusion and morphology in mammalian cells.

Design of a nonviral vector for site-selective, efficient integration into the human genome.

Gene therapy in eukaryotes has met many obstacles. Research into the design of suitable nonviral vectors has been slow. To our knowledge, no nonviral vector has been proposed that allows for the possibility of highly efficient, site-selective integration into the genome of mammalian cells. On the basis of prior studies investigating the components necessary for transposon, retrovirus-like retrotransposon, and retroviral integration, we propose a nonviral system that would potentially allow for site-selective, efficient integration into the mammalian genome. Transposons have been developed that can transform a variety of cell lines. For example, the Sleeping Beauty transposon (SB) can transform a wide range of vertebrate cells from fish to human, and it mediates stable integration and long-term transgene expression in mice. However, the efficiency of transposition varies significantly among cell lines, suggesting the possible involvement of host factors in SB transposition. Here, we propose the use of a chimeric transposase (i.e., transposase-host DNA binding domain) to bypass the potential requirement of a host DNA-directing factor (or factors) for efficient, site-selective integration. We also discuss another potential method of docking the transposon-based vector adjacent to the host DNA, utilizing repetitive sequences for homologous recombination to promote efficient site-selective integration, as well as other site-selective nonviral approaches.

A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect-inducing, helper-dependent E1-positive particles.

An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6. This E1-transformed human cell line contains only Ad5 nucleotides 459-3510, which precludes double crossover-type homologous recombination because the Ad5FGF-4 only contains 5' Ad5 sequence up to nucleotide 453. The Ad5FGF4 vector does, however, retain 177 nucleotides of the 3' end of the E1B-55K gene that are also present in PER.C6. With only this single region of homology between vector and cell line, we were surprised to detect virus-specific cytopathic effects (CPE) in our cell-based assay for RCA. This CPE-inducing agent was amplified in nonpermissive A549 cells but also supported amplification of the parental Ad5FGF-4. Because we were unable to isolate the CPE-inducing agent in pure form we first identified it as atypical RCA. Polymerase chain reaction (PCR) and Southern blot experiments identified viral DNA segments in which recombination had occurred between the 177 nucleotides of E1B present in both Ad5FGF-4 and PER.C6. The atypical RCA genomes contain a copy of the original (PGK promoter-E1 gene carrying) plasmid used in the construction of the PER.C6 cell line and they retained the parental FGF-4 transgene. However, significant deletions occurred within the recombined genomes in compensation for the large insertion from PER.C6 sequences and resulted in the loss of essential viral genes. This deletion renders these recombinant viruses replication defective, requiring helper functions from remaining parental Ad5FGF-4 for amplification. These atypical RCA entities may be more properly designated as helper-dependent E1-positive particles (HDEPs). This finding shows the importance of avoiding the use of "nonmatched" vectors where any overlap exists between the recombinant vector and E1 sequences in the packaging cell line. The cloning of the FGF-4 transgene into an adenoviral vector specifically "matched" for PER.C6 (lacking the 177 nucleotide region of homology) has allowed extensive virus propagation (Ad5.1FGF-4) with no CPE- or HDEP-like events yet detected.

Evidence for Repeat-Induced Gene Silencing in Cultured Mammalian Cells: Inactivation of Tandem Repeats of Transfected Genes

Foreign DNA can be readily integrated into the genomes of mammalian embryonic cells by retroviral infection, DNA microinjection, and transfection protocols. However, the transgenic DNA is frequently not expressed or is expressed at levels far below expectation. In a number of organisms such as yeast, plants, Drosophila, and nematodes, silencing of transfected genes is triggered by the interaction between adjacent or dispersed copies of genes of identical sequence. We set out to determine whether a mechanism similar to repeat-induced gene silencing (RIGS) is responsible for the silencing of transgenes in murine embryonal carcinoma stem cells. We compared the expression of identical reporter gene constructs in cells carrying single or multiple copies and found that the level of expression per integrated copy was more than 10-fold higher in single-copy integrants. In cells carrying tandem copies of the transgene, many copies were methylated and clones frequently failed to express both copies of near-identical integrated alleles. Addition of extra copies of the reporter gene coding sequence reduced the level of expression from the same reporter driven by a eukaryotic promoter. We also found that inhibitors of histone deacetylase such as trichostatin A forestall the silencing of multicopy transgenes, suggesting that chromatin mediates the silencing of transfected genes. This evidence is consistent with the idea that RIGS does occur in mammalian embryonic stem cells although silencing of single-copy transgenes also occurs, suggesting that RIGS is only one of the mechanisms responsible for triggering transgene silencing.

Simple and efficient vectors for retrofitting BACs and PACs with mammalian neoR and EGFP marker genes

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are widely used to investigate the functions of genes and genomes in mammalian cells invitro and invivo. We have developed a series of vectors which can simply and efficiently be retrofitted onto BACs or PACs. These vectors carry a neoR gene for selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick detection of the DNA in cells. All the plasmids are retrofitted using the loxP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitting of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be monitored by the EGFP fluorescence.

Camptothecin enhances random integration of transfected DNA into the genome of mammalian cells

In order to study the involvement of DNA topoisomerase I (top1) in recombination, we examined the effect of the anti-neoplastic drug camptothecin, which selectively poisons top1 by trapping top1-cleavable complexes on integration of exogenic vector into the genome of mammalian cells. We transfected mouse F9 teratocarcinoma cells as well as Chinese hamster V79 cells with a plasmid carrying a selectable neo gene treated with camptothecin, and determined the frequency of neo+ (G418 R) colonies. We found that treatment with camptothecin for as short a time as 4 h after electroporation resulted in a 4- to 33-fold stimulation of plasmid integration into the recipient genome via non-homologous recombination. These results imply that top1-cleavable complexes trapped by camptothecin could be potentially recombinogenic structures and could stimulate non-homologous recombination in vivo, promoting the integration of transfected plasmids into mammalian genome.