Pierluigi Donini

Genetically modified pigs produced with a nonviral episomal vector

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

The number of vertebrate repeats can be regulated at yeast telomeres by Rap1‐independent mechanisms

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1‐independent mechanism regulates the number of yeast telomeric repeats (TG1–3) and of vertebrate repeats (T2AG3) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T2AG3‐only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1‐independent manner, both in TEL1 and in tel1Δ humanized yeast cells. Altogether, these findings shed light on multiple repeat‐counting mechanisms, which may share critical features between lower and higher eukaryotes.

Functional human CFTR produced by a stable minichromosome.

Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.

Molecular and cytological analysis of a 5.5 Mb minichromosome

Mammalian artificial chromosomes (MACs) provide a new tool for the improvement of our knowledge of chromosome structure and function. Moreover, they constitute an alternative and potentially powerful tool for gene delivery both in cultured cells and for the production of transgenic animals. In the present work we describe the molecular structure of MC1, a human minichromosome derived from chromosome 1. By means of restriction and hybridization analysis, satellite‐PCR, in situ hybridization on highly extended chromatin fibres, and indirect immunofluorescence, we have established that: (i) MC1 has a size of 5.5 Mb; (ii) it consists of 1.1 Mb alphoid, 3.5 Mb Sat2 DNA, and telomeric and subtelomeric sequences at both ends; (iii) it contains an unusual region of interspersed Sat2 and alphoid DNAs at the junction of the alphoid and the Sat2 blocks; and (iv) the two alphoid blocks and the Sat2‐alphoid region bind centromeric proteins suggesting that they participate in the formation of a functional kinetochore.

Mammalian artificial chromosomes — vectors for somatic gene therapy

Mammalian artificial chromosomes might prove to be useful vectors for somatic gene therapy. The functional elements of such an artificial chromosome are telomeres, a centromere and a replication origin. Recent progress in the characterization of these functional elements of the eukaryotic chromosome will be described. Attempts to construct artificial chromosomes for mammalian cells and their use for somatic gene therapy are discussed.

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli‐yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re‐arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.

Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig

A de novo SAC was constructed by making use of YAC technology and a humanized yeast strain. The construct (about 310 kb) contained pig centromeric DNA and the Neo gene. The construct was introduced into a pig cell line by yeast-mammalian cell fusion and G418 resistant clones were obtained. One clone was characterized by FISH and shown to contain an episomally located microchromosome containing YAC, Neo and pig centromere sequences. FISH analysis over time showed that the SAC was mitotically stable for at least 34 generations in the absence of selection. The size of the SAC was determined by confocal microscopy of the SAC and shown to be approximately 7 Mb, which is about 25-fold greater than the size of the original YAC. From its behavior in pulsed field gel electrophoresis, FISH analysis of stretched DNA fibers, and its appearance under scanning confocal microscopy, it was concluded that the SAC is a circularized and multimerized derivative of the original YAC. Possible applications as vectors for animal transgenesis are discussed.

In vitro identification of a protein of Saccharomyces cerevisiae that interacts specifically with the G-rich DNA strand of the telomere

The telomeres of Saccharomyces cerevisiae consist of a repeated G2-3T(GT)1-6 DNA sequence that forms a complex with proteins. To date only the RAP1 protein has been shown to bind to the simple sequences in yeast telomeric DNA, as well as to non-telomeric regulatory sites. We have used synthetic oligodeoxyribonucleotides, both double- and single-stranded, to identify specific yeast telomeric proteins in a partially purified yeast extract. Using the gel shift assay, we detected a binding activity that is stable at high ionic strength and that recognizes specifically the G-rich protrusion of a double-stranded synthetic yeast telomere, as well as the G-rich single strand. This is the first evidence of a purely telomeric protein in that it binds to the single-stranded telomeric protrusion of the yeast chromosome.

A human DNA telomeric fragment contains yeast ars and mitotic stabilizing sequences

A minilibrary of human DNA fragments was prepared in the vector YIP5 from a DNA preparation enriched for telomeric sequences. Screening of the library produced one clone that hybridized to the TTAGGG sequence. The cloned DNA fragment was shown to be telomeric by a number of criteria. In situ hybridization to metaphase human chromosomes showed that the fragment hybridized to the tips of all human chromosomes. The fragment contained at least two yeast autonomously replicating sequences (ARS) and stabilizing sequences, since it transformed Saccharomyces cerevisiae with high efficiency, giving rise to clones which were mitotically stable under non-selective growth.

Functional telomere formation in yeast using synthetic C4A2 sequences.

A yeast artificial chromosome (YAC) was constructed with a native autonomous replicating sequence (ARS) flanked telomere at one end and a 50-bp synthetic oligonucleotide of C4A2 repeats at the other. This was done in order to determine whether the presence of the flanking ARS sequence is required for telomere function. This construct was introduced into two different yeast strains: one mutated in the recombination function RAD52 and the other wild type for this gene. Both strains gave rise to autonomously replicating artificial chromosomes. The molecules in the RAD52 strain were rearranged dimers terminating at both ends with Tetrahymena telomeres, whereas in the rad52 strain two classes of YACs were found: rearranged dimers and elements bearing an ARS-free telomere. The presence of the latter class of molecules confirmed the finding of Wellinger and Zakian (1989, Proc. Natl. Acad. Sci. USA 86, 973-977) that the flanking ARS is not required for telomere function. Furthermore, in this class of molecules the ARS-free telomeric end was shortened as a result of deletions that removed some distal pBR322 sequences and some C4A2 repeats. The size of the resulting YACs ranged from 7.7 to 9 kb, considerably below the size threshold found by Zakian et al. (1986, Mol. Cell. Biol. 6, 925-932) for CEN4 artificial plasmids. An explanation for the structural instability of the ARS-free end of the YACs is suggested.