3.6-KB mouse cyclin C promoter fragment is predominantly active in the testis

Cyclin C is a highly conserved protein that regulates cell-cycle, messenger RNA transcription and cell adhesion. Recently published studies demonstrate that this protein is an essential player during early embryonic development of multicellular eukaryotes as well. In order to understand better its complex function at the level of tissues or organs, spatial expression characteristics of cyclin C and regulatory components of its expression are needed to be determined. In vitro studies on human cells suggested that approximately the first 3 kilobases of the cyclin C promoter might contain all the regulatory elements that might mimic transcription of cyclin C. To test the hypothesis, we generated reporter transgenic lines where the first 3.6-kilobase region of mouse cyclin C promoter fragment drives the transcription of a marker gene. Messenger RNA levels of the marker gene and cyclin C isoforms were measured in nine organs with reverse transcription coupled quantitative realtime polymerase chain reaction and their expression patterns were compared. The marker gene is predominantly transcribed in testes and does not follow the transcriptional regulation of the examined cyclin C isoforms. Thus, the isolated promoter fragment alone is not sufficient for the complete physiological modulation of cyclin C RNA levels, however, it is capable of enhancing testicular transcription which can be exploited in future applications

Novel method to load multiple genes onto a mammalian artificial chromosome.

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future

The assembly of the pSTRFP plasmid.

<p>(A) The pSEV2 entry vector was digested with the BamHI and HindIII restriction enzymes. (B) The pmR-mCherry plasmid was digested with the BamHI and HindIII restriction endonucleases, the RFP CDS-containing DNA fragment was isolated and inserted into the pSEV2 plasmid and the pS2RFP vector was assembled. (C) The I-Ceu and PI-PspI enzymes were used to transfer the mCherry expression cassette from pS2RFP into the pST plasmid and the pSTRFP ACE targeting vector was produced.</p

The pST plasmid vector was produced to achieve the superloading of Platform ACEs chromosomes.

<p>(A) The pSEV1R (shuttle expression vector-1R) entry vector map. The gene of interest is cloned into the multi-cloning site (MCS) of this vector. MCS is part of an expression cassette with a chicken beta-actin promoter and an SV40 polyA signal. The I-Ceu and PI-PspI restriction enzyme recognition sites flank the expression cassette. The whole expression cassette with the gene of interest can be moved from this plasmid into several different types of Platform ACEs targeting plasmids (ATVs) with the help of these enzymes. (B) A Platform ACEs-targeting plasmid, pATVMin. The I-Ceu and PI-PspI enzyme sites are used to transfer the expression cassette with the gene of interest into pATVMin. The attB site is the recognition site of ACE integrase. This enzyme performs site-specific recombination between the attB site and the attP site found on Platform ACEs. This process integrates the pATVMin plasmid together with the gene of interest in the expression cassette into the artificial chromosome. After integration, the promoterless neomycin resistance gene acquires the promoter of puromycin gene on the Platform ACEs and G418-resistant cell lines can be isolated with ACE chromosomes carrying and expressing the gene of interest. (C) The pmccKO14 plasmid map. The HSV-TK expression cassette with the LoxP site was removed from this plasmid by XbaI-SpeI restriction enzyme digestion and transferred into the pATVMin plasmid. (D) The map of the pST plasmid. This plasmid contains a promoterless neomycin gene and a HSV-TK expression cassette flanked by direct repeats of two LoxP sites. The HSVT-TK expression cassette came into this plasmid from the pmccKO14 vector by ligation into the unique XbaI site in the pATVMin plasmid. The LoxP site upstream from NeoR CDS was inserted by a PCR-based method. The gene of interest is transferred into the pST vector by the method described in (B). This transgene-carrying vector is then loaded onto the Platform ACEs chromosome as described in (B) and previously published⁷. The LoxP-flanked selectable marker gene cassette can be removed by the transient action of Cre recombinase. Through the use of ganciclovir selection, cell lines lacking the neomycin-thymidine kinase selectable marker gene cassette can be isolated.</p

The “superloading” cycle.

<p>The transgene is cloned into the pST plasmid and loaded onto the ACE chromosome as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085565#pone-0085565-g001" target="_blank">Figure 1</a>. In the third step the Cre recombinase is transiently expressed in the cell line carrying the transgene-loaded ACE. The Cre recombinase removes the neomycin-thymidine kinase selectable marker gene cassette found between the direct repeats of LoxP sites. Transgene-loaded ACE chromosome-carrying cell lines that lack the neomycin-thymidine kinase selectable marker gene cassette can be obtained by ganciclovir selection. The ACE chromosome is now suitable for the loading of a new transgene cloned into the pST plasmid vector.</p