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.

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.

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.

Chromosome engineering with lambda-integrase mediated recombination system: the ACE system.

Mammalian satellite DNA-based artificial chromosomes (SATACs) are unique among the mammalian artificial chromosomes. These reproducibly generated de novo chromosomes are stably maintained in different species, readily purified from the host cell's chromosomes and can be introduced into a variety of recipient cells. An artificial chromosome expression system (ACE system) has been developed on these SATACs to extend them for chromosome engineering. This system includes a Platform ACE containing multiple acceptor sites, specially designed targeting vector (ATV), and an ACE-integrase expression vector (pCXLamIntROK). Gene of interest are cloned into targeting vector (ATV), and site-specific loading of genes onto Platform ACE is facilitated by ACE-integrase mediated recombination. ACE system is suitable for multiple or subsequent loading of useful genes onto the same chromosome vector. This chapter describes the detailed procedure of chromosome engineering using the ACE system.

Alignment of biological microparticles by a polarized laser beam.

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581-1590, 2003) have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic light-harvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes.

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale ( approximately 100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (PsiNF1) multisequence. The two DNA types are joined by an approximately 2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning approximately 200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4-PsiNF1 DNA segment appeared first on the ancestor of human chromosome 17.