Genome Of Recipient Cells Research Articles

Extracellular vesicles mediate the horizontal transfer of an active LINE-1 retrotransposon

ABSTRACTLong interspersed element-1 (LINE-1 or L1) retrotransposons replicate through a copy-and-paste mechanism using an RNA intermediate. However, little is known about the physical transmission of retrotransposon RNA between cells. To examine the horizontal transfer of an active human L1 retrotransposon mediated by extracellular vesicles (EVs), human cancer cells were transfected with an expression construct containing a retrotransposition-competent human L1 tagged with a reporter gene. Using this model, active retrotransposition events were detected by screening for the expression of the reporter gene inserted into the host genome by retrotransposition. EVs including exosomes and microvesicles were isolated from cells by differential centrifugation. The enrichment of L1-derived reporter RNA transcripts were detected in EVs isolated from cells expressing active L1 retrotransposition. The delivery of reporter RNA was confirmed in recipient cells, and reporter genes were detected in the genome of recipient cells. Additionally, employing qRT-PCR, we found that host-encoded factors are activated in response to increased exposure to L1-derived RNA transcripts in recipient cells. Our results suggest that the horizontal transfer of retrotransposons can occur through the incorporation of RNA intermediates delivered via EVs and may have important implications for the intercellular regulation of gene expression and gene function.

Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.

The role of tumor-derived exosomes in epithelial mesenchymal transition (EMT).

Exosomes are membrane-bound small vesicles (30–150 nm) produced by all cell types and present in all body fluids (1). They are a part of the intercellular communication system that is evolutionarily conserved and operates in bacteria as well as all multicellular organisms (2). Tumor cells produce and release masses of exosomes into the extracellular space. These exosomes carry information in the form of molecular signals and/or genetic materials (mRNA, miRNA, DNA) from the parent tumor cell to locally- or distantly-located recipient cells. Exosome-mediated transfer of information results in re-programming of the recipient cell genome and proteome and ultimately leads to the acquisition of new cellular functions (3).

Rhodobacter capsulatus DprA is essential for RecA-mediated gene transfer agent (RcGTA) recipient capability regulated by quorum-sensing and the CtrA response regulator.

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.

Protein apparatus for horizontal transfer of agrobacterial T-DNA to eukaryotic cells

This review analyzes agrobacterial virulence proteins and recipient cell proteins involved in horizontal transfer of a T-DNA-protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange, attachment, close contact); T-DNA release from bacterial cells; channel formation for the transfer of ssDNA between the partners; transfer of agrobacterial T-DNA through the membrane, cytoplasm, and nuclear membrane of the recipient cell and its incorporation into the recipient cell genome. It further discusses possible pathways of agrobacterial ssDNA transfer to the recipient cells. In particular, the possible role of T-pili and VirE2 protein during conjugative transfer of agrobacterial ssDNA between donor and recipient cells is discussed.

Donor DNA Can Be Detected in Kidney Recipient Blood for Years after Kidney Transplantation Using Forensic Medicine Methods

After transplanted organ revascularization, DNA from passenger cells and cellular debris from ischemic and rejected cells is disseminated in the recipient. The question is whether this DNA is disintegrated or stored in scavenger cells or be incorporated into recipient cell genome in the process of the so called “horizontal or illegitimate DNA incorporation” a frequent phenomenon in nature preponderantly in bacteria. Aim. To search for donor DNA in recipient tissues and cells after allogeneic transplantation and immunosuppression. Materials: Recipient's blood samples were collected before and after kidney transplantation 1, 14,28,90, 180, 360 and 720 days and genomic DNA was isolated. Short tandem repeats analysis (STR) was applied. The investigated loci were: phospholipase A2-HUMPLA2A1(AAT)n, cytochrome P450 HUMCYARO(AAAT)n and D1S80. Results: Donor cytochrome P450 or HUMPLA2 or D1S80 genes were detected in recipient blood cells up to 2 years after kidney transplantation. Positive results were observed 24hr after grafting in 3 out of 3pts and in 4 out of 4 pts after 14 days, in 28 out of 28 after 28, in 2 out of 2after 90, in 3 after 180, in 1 after 1 and in 2 out of 2 after 2 years. In 2 cases recipient blood cell nuclei were isolated and donor A2- HUMPLA2A1(AAT)n was detected. Conclusions: Donor DNA can be detected in recipient blood cells 2 years after kidney transplantation. The question as to whether the detected donor DNA was contained in the surviving donor cells or in a form of apoptotic or necrotic bodies in recipient phagocytes or was incorporated into recipient cell genome remains to be answered. The presence of donor DNA in recipient blood cell nuclei indicates that it is still present in surviving passenger cells or has in some way been incorporated into recipient cell genome. This last is quite likely as in animal experiments passenger cells can not be detected aslong as 2 years.

The Influence of Donor Dna Level in Blood of Simultaneous Pancreas-Kidney Allograft Recipients as a Diagnostic Factor for Rejection

Introduction: Grafted organ releases into circulation system of recipient some cells, DNA and proteins which are partially destroyed by the recipient's phagocytes but they can be also detected in spleen dendritic cells. The aim of our research was to study the possible effects of microchimerism in pancreas and kidney rejection. In our previous studies, we looked for the presence of donor-specific STR loci (phospholipase A2-HUMPLA2A1(AAT)n, cytochrome P-450 A2-HUMCYARO(AAAT)n and locus D1S80) in the recipient blood. Methods: In this study we have examined plasma and mononuclear whole blood cells of recipients to assess the amount of SRY gene in sexmismatched combinations and phospholipase A2-HUMPLA2A1(AAT) n in the same sex recipients after simultaneous pancreas and kidney transplant, in relation to the patients probes before grafting by the use of the Real-time PCR method. Recipient's blood and donors spleen samples were collected before transplantation and at different times after grafting. Genomic DNA was isolated from blood and tissues. The amount of DNA in different samples after tx was calculated by using the comparative Ct method with GAPDH as internal control. Results: We observed increase donor's DNA level in recipient's blood already on day 1 after grafting (STR loci). The quantitative indication of donor DNA in recipient's blood (SRY gene) was possible in 7 day after graft. In case of HUMPLA gene, our results showed that the relative amount of that gene was higher in mononuclear blood cells than in plasma 14 days after transplantation. Conclusion: Donor DNA is present either in passenger cells or recipient's phagocytes. An open question remains whether it may be incorporated into recipient cell genome and what is the impact on tolerance development.

Approaches to site-directed DNA integration based on transposases and retroviral integrases

The ability of retroviruses and transposons to insert their genome into the host cell makes them attractive objects for constructing gene therapy vectors. However, enzymes that insert genetic material do not possess any selectivity relative to target nucleotide sequences, which results in almost random DNA insertion into the recipient cell genome. This leads to mutations that in turn may cause certain undesirable consequences and sometimes neoplastic cell transformation. For successful functioning, it is a primary necessity to modify a retrovirus and transposon based genetic therapy systems in such a way that the directed vector integration into a target sequence in the human genome can be achieved. In this review, the approaches to date that have been developed for highly specific modification of the genome using fusion protein construction based on retroviral integrases and transposases are discussed, as well as cellular factors interacting with these enzymes.

Development and Optimization of Method for Generating Unmarked <I>A. tumefaciens</I> Mutants*

Agrobacterium tumefaciens possesses many advantages as a model bacterium for the study of a wide variety of biological processes. Gene disruption or inactivation is a powerful and direct tool for investigation of in vivo gene functions. The intensive study of A. tumefaciens has increased the need for simple and highly efficient procedures to manipulate its genome. The sacB gene was used as a counterselectable marker to develop a gene replacement procedure that allows precise insertion, deletion, and allele substitution of any gene sequence in A. tumefaciens without altering the genome in any other way. A kanamycin resistance (Km(superscript R) cassette was constructed to the suicide vector as the positive selection marker. The suicide plasmid containing DNA fragments homologous to the flanking sequences of the target gene was integrated into the recipient cell genome at the target gene locus by intermolecular homologous recombination, generating the Km(superscript R)-single cross-over colonies. The effect of homologous sequence length on the intermolecular homologous recombination was analyzed. The second cross-over colonies generated by intermolecular homologous recombination occurring between two tandem repeats were simply screened out by counter-selection of sacB. Data showed that the intervening sequence length between two repeats significantly affected the intermolecular homologous recombination frequency in A. tumefaciens, indicating that A. tumefaciens adopted the homologous recombination mechanism similar to that in E. coli. All these results demonstrated that investigators could minimize the numbers of colonies to be analyzed and reduce the overall workload by optimizing the relative length of two homologous fragments and using the specific type of single cross-over transformants for screening the second cross-over event. This mutagenesis strategy had successfully been used to generate the double unmarked △vbp2△vbp3 mutant in two A. tumefaciens strains.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Qualitative and quantitative characteristics of the extracellular DNA delivered to the nucleus of a living cell

BackgroundThe blood plasma and other intertissue fluids usually contain a certain amount of DNA, getting there due to a natural cell death in the organism. Cells of this organism can capture the extracellular DNA, whereupon it is delivered to various cell compartments. It is hypothesized that the extracellular DNA is involved in the transfer of genetic information and its fixation in the genome of recipient cell.ResultsThe existence of an active flow of extracellular DNA into the cell is demonstrated using human breast adenocarcinoma (MCF-7) cells as a recipient culture. The qualitative state of the DNA fragments delivered to the main cell compartments (cytoplasm and interchromosomal fraction) was assessed. The extracellular DNA delivered to the cell is characterized quantitatively.ConclusionIt is demonstrated that the extracellular DNA fragments in several minutes reach the nuclear space, where they are processed so that their linear size increases from about 500 bp to 10,000 bp. The amount of free extracellular DNA fragments simultaneously present in the nuclear space may reach up to 2% of the haploid genome. Using individual DNA fragments with a known molecular weight and sequence as an extracellular DNA, it is found that these fragments degrade instantly in the culture liquid in the absence of a competitor DNA and are delivered into the cell as degradants. When adding a sufficient amount of competitor DNA, the initial undegraded molecules of the DNA fragments with the known molecular weight and sequence are detectable both in the cytoplasm and nuclear space only at the zero point of experiments. The labeled precursor α-dNTP*, added to culture medium, was undetectable inside the cell in all the experiments.

Evidence for horizontal transfer of the LTR retrotransposon mdg3, which lacks an env gene.

Horizontal (interspecific) transfer is regarded as a possible strategy for the propagation of transposable elements through evolutionary time. To date, however, conclusive evidence that transposable elements are capable of horizontal transfer from one species to another has been limited to class II or DNA-type elements. We tested the possibility of such transfer for several Drosophila melanogaster LTR retrotransposons of the gypsy group in an experiment in which D. melanogaster and D. virilis somatic cell lines were used as donor and recipient cells, respectively. This approach was chosen in light of the high levels of LTR retrotransposon amplification and expression observed in cultured D. melanogaster cells. In the course of the experiment, parallel analysis for mdg1, mdg3, 17.6, 297, 412 and B104/roo retrotransposons was performed to detect their presence in the genome of recipient cells. Only the mdg3 retrotransposon, which lacks an env gene, was found to be transmitted into recipient cells. This model, based on the use of cultured cells, is a promising system for further investigating the mechanisms of LTR retrotransposon transfer.

Synthetic retrotransposon vectors for gene therapy.

New gene therapy methods are rapidly being developed to permit the expression of tumor suppressor genes, cytotoxins, anticancer antigens, and immunoregulatory proteins in the treatment of cancer. Large-scale testing in humans has been delayed by questions concerning the safety and effectiveness of preferred retroviral vectors and helper cells. These vector systems are limited by their ability to undergo homologous recombination with endogenous retroviruses or helper-viral sequences, resulting in release of replication-competent retrovirus (RCR). In addition, transcriptional inactivation of the retroviral promoter often occurs, caused in part by methylation of CpG islands in the retroviral long terminal repeats (LTRs). We report the production of highly specific retrovectors using gene amplification together with oligonucleotide building blocks. The synthetic vectors were based on mouse VL30 retrotransposon NVL3, and lacked homology to retroviral helper gene sequences. Three of four constructs made by gene amplification yielded biologically active vectors. These constructs efficiently transmitted and stably inserted a neomycin resistance marker gene into the genome of recipient cells, expressing an abundant RNA species of the expected size in the absence of detectable replication competent retrovirus. The vectors and techniques described enable widely applicable expression modes using generic helper cells, and require only approximately 1.3 kb of cis-acting vector RNA sequences for faithful transfer and expression of genetic material.

DNA repair and the evolution of transformation in Bacillus subtilis. III. Sex with damaged DNA.

Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. We document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome.

A retrovirus vector which transduces a functional estrogen receptor gene at high efficiency.

A high titer retroviral vector containing the cDNA of human estrogen receptor (hER) was generated and used to transfer the hER gene into the rat 208F cell line. Southern blot analysis showed the integration of the provirus to be at a unique site and that the provirus was intact in the genome of recipient cells. The expression of the integrated hER gene in the infected rat cells was detected by Northern blot analysis and by a functional assay in which the hER gene product stimulated the production of a chloramphenicol acetyl transferase gene under the control of an estrogen-responsive element. These experiments demonstrate the feasibility of using a retroviral vector system to introduce a functional ER gene into cultured cells lacking this receptor.

Fifty years on: molecular biology's hall of fame

Genetic material is absorbed onto the surface of minute tungsten beads. The beads are then explosively discharged against plant tissue and, in some instances, penetrate individual cells. Some cells are stably transformed and the GUS gene has been expressed after such treatment. This method of transformation does not rely on sophisticated plant protoplast systems and the species to be transformed need not be susceptible to biological transforming agents such as Agrobacterium tumefaciens. The chief requirements for transformation are that the bombarded cells be regenerable and that the inserted genetic material be compatible with the recipient cell genome. Thus, particle bombardment is potentially significant as a 'generic' approach to plant transformation, having application to a wide range of species and, generally, requiring readily available regeneration systems (i.e. from tissue). Synthetic seeds The plant division meeting ended with a special symposium on synthetic seeds. Synthetic seed technology seeks to use somatic embryos as high efficiency clonal propagules. Synthetic seeds would be useful for clonal production of hybrid strains, for the rapid multiplication of genetically engineered seed-propagated crops and for germplasm preservation, particularly for clonally propagated plants. One major line of research involves conferring desirable seed-like qualities, such as the existence of a reversible dehydrated resting phase, on somatic embryos. McKersie et al. (University of Guelph, Guelph) reported that plants could be regenerated from alfalfa somatic embryos stored in a dehydrated state at ambient temperatures for up to eight months. Such a quiescent resting phase will be necessary for synthetic seed of most crops to be commer-

Enhanced transforming activity of ultraviolet-irradiated pSV2- gpt is due to damage outside the gpt transcription unit

We have shown that when pSV2- gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt + transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2- gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m 2, but remained relatively constant at doses between 2 and 8 kJ/m 2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of the gene.

A vehicle for DNA transfer and for recovery of transferred genes: λ Charon phage-pBR322 hybrid

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase ( tk) gene, the ampicillin-resistance (Ap R) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse L tk − cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the L tk − transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the L tk + cell into Escherichia coli; the method depends on the fact that the recombinant 2 phage carrying the Ap R gene and replication origin ofpBR322 transduces λ-lysogenic bacteria to Ap R and is maintained as a plasmid. Using this method the HSV-1 tk gene from one L tk + transformant was rapidly and successfully recovered without any rearrangement of the target sequence.

Transformation by subgenomic fragments of Rous sarcoma virus DNA

Subgenomic fragments of Rous sarcoma virus (RSV) DNA, generated by Eco RI digestion of DNA of RSV-infected chicken cells, induced transformation of NIH/3T3 mouse cells with efficiencies that were 100–1000 fold lower than the efficiency of transformation by intact RSV DNA. Analysis of the DNAs of NIH cells transformed by Eco RI-digested RSV DNA indicated that these cells contained no more than 2 × 10 6 daltons of RSV DNA, and did not contain sequences from the 5′ terminus of RSV RNA which are included in the leader sequence of subgenomic src mRNA of RSV-infected cells. The product of the RSV src gene (pp60 src), however, was produced in apparently similar quantities by NIH cells transformed by Eco RI fragments of RSV DNA and by intact RSV DNA. Thus expression of the src gene of RSV in NIH cells transformed by subgenomic fragments of RSV DNA did not require the terminal sequences of the RSV genome, which appear to be involved in synthesis and processing of src mRNA in RSV-infected cells. DNAs of NIH cells transformed by Eco RI-digested RSV DNA were found to induce transformation in secondary transfection assays with efficiencies that were similar to the efficiency of transformation by intact RSV DNA. These results suggest that transformation by subgenomic fragments of RSV DNA may be a consequence of integration of src gene-containing DNA fragments in the vicinity of a promoter site in the recipient cell genome, leading to efficient expression of the RSV src gene.

Hypothesis on the significance of RNA-directed DNA synthesis for evolution.

The hypothesis is advanced that genetic information packaged in protoviruses is routed both horizontally (from cell to cell and from organism to organism) and vertically (from somatic cells to germ cells) as a means of advancing evolution. Protoviruses are produced in response to environmentally-induced stress and via the RNA → DNA feedback loop are inserted into the genome of recipient cells. Once inserted there, they confer new biochemical properties to the host cell which are normally of selective value.