Are there plasmids in eukaryotic cells

Try out PMC Labs and tell us what you think. Learn More. Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu extra chromosomal unit , for DNA delivery using L.

This plasmid exhibits the following features: 1 a theta origin of replication and 2 an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus pCMV. The functionality of pExu: egfp was evaluated by fluorescence microscopy.

The L. The pExu vector has demonstrated an excellent stability either in L. The eGFP expression at different times in in vitro assay showed that The enterocytes of mice showed the expression of eGFP protein. Thus, L. Lactic acid bacteria LAB have been reported to be useful for mucosal delivery of different molecules, like heterologous proteins, vaccine, and plasmids. Lactobacilli, lactococci, enterococci, streptococci, leuconostoc, and pediococci are the broad genera of these bacterial group; habitats, morphology, optimum temperature, pH and salt tolerance, and pathogenic potential are characteristics in which they differ.

These Gram-positive bacteria are non-pathogenic and non-invasive and inhabit different ecological niches plant surfaces and the digestive tract of animals and human. The strategy of bactoinfection live bacterial vectors for transfection of mammalian cells opened the field to use L. Here, we developed a new plasmid called pExu extra chromosomal unit for DNA delivery. To construct this new plasmid, we used as backbone pOri, a shuttle E. The eukaryotic region, derived from pCDNA3.

After oral administration of L. The shuttle pExu plasmid 6. The new plasmid was successfully stabilized in E. Not to scale. This shuttle-cloning vector was successfully introduced into E.

As well documented by Rocha and colleagues, 18 L. Thereby, different strains of genetically modified GM LAB, with inherent anti-inflammatory, can have this characteristic increased and also new ones can be incorporated. Structural and segregational analysis in E. Left E. The assays of selective pressure were done during 5 days generations for E. The structure of pExu: egfp was confirmed by PCR, enzymatic digestion, and sequencing.

CHO cells were transfected with pExu: egfp. The confocal microscopy showed the expression of eGFP protein by eukaryotic cells Figure 3. Also, the kinetic analysis by fluorescent microscopy after 6, 12, 24, 48, and 72 hr post-transfection showed that eGFP was expressed from 12 hr until at least 72 hr Figure 4. The cells were incubated with DAPI for nuclear staining. The data were analyzed using the software BD CFlow. The oral administration of L.

It was not possible to detect eGFP expression until 6 hr and after 72 hr post gavage Figure 6. Furthermore, we were not able to detect expression of eGFP protein by eukaryotic cells in the ileum portion at the same times points data not shown.

Genetically modified LAB can be used as vectors for local delivery of biologically active molecules protein or DNA either to the gastrointestinal tract or other mucosal surfaces improving the targeting of recombinant antigens.

Since L. In , our group developed a plasmid to be used in E. In , Tao and colleagues 30 showed by in vitro assays that the treatment with glycine, which is able to fragilize the cell wall of L. Nonetheless, it was reported that integrity of the whole membrane structure is essential for fruitful delivery of DNA in eukaryotic cells. Another intelligent approach using L. This strategy allowed the possibility to clone the gene of interest in frame with a reporter gene and to evaluate the expression of the target gene by simple observation of the reporter.

They did not use any chemical enhancers. In fact, this plasmid has an RCR origin; it is not considered to have such a high stability as a plasmid with theta origin. However, they did not test its functionality in in vivo experiments. Moreover, as we showed in this report, the results can be different due to different environments and conditions. In this report, we presented a new shuttle vector, called pExu, to be used in native L.

This vector is suitable for use in E. Our results confirmed that the pExu plasmid carried either by E. There were no structural changes after and hr for E.

Moreover, this plasmid has shown a high segregational stability. Then, we cloned the egfp ORF successfully in the pExu vector. The confocal analysis, as well as flow cytometry performed in this study with transfected CHO cells, revealed the functionality of the pExu: egfp vector.

The mammalian cells were capable of producing the reporter protein. Our images obtained by confocal or fluorescence microscopy after transfection showed a low expression of eGFP protein by mammalian cells. We awarded these results to the large size of the new plasmid 6, bp added to the reagent for transfection applied to in vitro assays. Although the eGFP expression was low in in vitro tests, we decided to transform L.

The epithelial cells of the duodenal region of mice orally gavaged with L. It was not possible to see the expression of eGFP protein in epithelial cells of the ileum region and colon data not shown.

These results were not in accordance with Almeida and colleagues. Our results allowed to confirm that the oral administration of L. To show that the bacterial capture and the protein expression is a transient process, we evaluated the eGFP expression by the host cells from 6 to hr after mice gavage. We were able to see expression only between 12 and 72 hr after gavage, confirming the previous observation by Chatel and colleagues in Related to the 6 hr time point, it is likely a too short time for an eukaryotic cell to do the transcription, translation, and post-translational processing to express the eGFP protein in in vivo systems.

Nonetheless, the no expression in the 96 to hr is due to the cell extrusion process. The particularly short lifetime of IECs intestinal epithelial cells had shown a rebirth of the functional villus epithelium by the stem cells of the crypts every 2 to 6 days in the greatest adults mammals. Even more, the best turnover rates of a fixed-cell population in the body are the enterocytes. Although the vector has the erythromycin resistance marker, which is not approved by the FDA, this resistance marker could be exchanged by kanamycin as well as eliminated using auxotrophic systems.

The in vivo results here obtained encouraged us to test it in disease models as well as in gene therapy. Bacteria and plasmids used in this study are listed in Table 1.

DNA manipulation protocols were performed as described previously in Sambrook and colleagues 45 with slight modifications. For plasmid DNA extraction from L. Enzymes were used as recommended by suppliers. Transformation of L. Related Concepts You have authorized LearnCasting of your reading list in Scitable. Do you want to LearnCast this session? This article has been posted to your Facebook page via Scitable LearnCast. Change LearnCast Settings.

Scitable Chat. This review will not consider plasmids of prokaryotic origin, even though certain bacterial plasmids, such as the tumor-inducing Ti plasmids of Agrobacterium tumefaciens, may be intimately associated with transformation of the eukaryotic host. This book, moreover, does not consider transformation experiments in eukaryotic hosts involving viral DNA as vectors, although indeed such vectors have been developed for use in plant and animal systems.

After a general introduction, providing historical perspective on the nature and role of plasmids, a list of eukaryotic plasmids will be presented according to their origin.

This is followed by a detailed discussion of known structure and function. In subsequent chapters the practical implications of eukaryotic plasmids for molecular cloning and biotechnology will be discussed. This latter part traces the development of interest'in biotechnical genetics and gives special consideration to the use of eukaryotic systems for gene cloning.

Biotechnical genetics includes, but is not limited to, gene cloning through recombinant DNA technology.