Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells

Guiducci, Cristiana; Ascenzioni, Fiorentina; Auriche, Cristina; Piccolella, Enza; Guerrini, Anna Maria; Donini, Pierluigi

doi:10.1093/hmg/8.8.1417

Cited by 28 publications

(24 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Minichromosomes (all containing ␣ satellite repeats) of less than 2.5 Mb, thus, have been created. Finally, several authors explored the possibility of using naturally occurring minichromosomes (Raimondi et al 1996;Guiducci et al 1999).…”

mentioning

confidence: 99%

Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Voet¹,

Vermeesch²,

Carens³

et al. 2001

Genome Res.

View full text Add to dashboard Cite

A small accessory chromosome that was mitotically stable in human fibroblasts was transferred into the hprt − hamster cell line CH and developed as a human chromosomal vector (HCV) by the introduction of a selectable marker and the 3Ј end of an HPRT minigene preceded by a loxP sequence. This HCV is stably maintained in the hamster cell line. It consists mainly of alphoid sequences of human chromosome 20 and a fragment of human chromosome region 1p22, containing the tissue factor gene F3. The vector has an active centromere, and telomere sequences are lacking. By transfecting a plasmid containing the 5Ј end of HPRT and a Cre-encoding plasmid into the HCV + hamster cell line, the HPRT minigene was reconstituted by Cre-mediated recombination and expressed by the cells. The HCV was then transferred to male mouse R1-ES cells and it did segregate properly. Chimeras were generated containing the HCV as an independent chromosome in a proportion of the cells. Part of the male and female offspring of the chimeras did contain the HCV. The HCV + F1 animals harbored the extra chromosome in >80% of the cells. The HCV was present as an independent chromosome with an active centromere and the human F3 gene was expressed from the HCV in a human-tissue-specific manner. Both male and female F1 mice did transmit the HCV to F2 offspring as an independent chromosome with properties similar to the original vector. This modified small accessory chromosome, thus, shows the properties of a useful chromosomal vector: It segregates stably as an independent chromosome, sequences can be inserted in a controlled way and are expressed from the vector, and the HCV is transmitted through the male and female germline in mice.

show abstract

mentioning

confidence: 99%

Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Voet¹,

Vermeesch²,

Carens³

et al. 2001

Genome Res.

View full text Add to dashboard Cite

show abstract

“…So far, inter-species chromosome transfer was used to carry human chromosomes into mouse ES cells, chinese hamster cells or bovine fibroblasts (Tomizuka et al, 1997;Hernandez et al, 1999;Guiducci et al, 1999;Kuroiwa et al, 2002). The transfer of a HAC from a hamster cell line to a mouse ES cell line was reported by Paulis et al, (2007).…”

Section: Human Artificial Chromosomes and Microcell Mediated Chromosomentioning

confidence: 99%

Multi‐transgenic pigs for xenotransplantation

Fischer

Kraner-Scheiber

Flisikowski

et al. 2013

Xenotransplantation

View full text Add to dashboard Cite

Genetically modified pigs offer a solution to the severe shortage of organs available for human transplantation. Inactivation of the α1,3‐galactosyltransferase gene, responsible for α1,3‐Gal epitope synthesis (1–2) was a major breakthrough and essentially solved the problem of hyperacute rejection. However, further modifications of donor pigs are required to enable long term xenograft survival. These include expression of complement regulatory, antithrombotic, endothelium protective and immuno‐regulatory transgenes. Current evidence indicates that complement regulator transgenes, such as CD46 and CD55, must be expressed at least fivefold more than their human endogenous equivalent to effectively protect grafted tissue (3). To explore means of achieving high expression, we produced a series of BAC‐ and PAC‐vector constructs containing different sized genomic complement regulatory genes, from 70 to 190 kb, under the control of endogenous or CAGGs (CMV enhancer chicken β actin) promoter sequences. Using these vectors we obtained high levels of CD46 and CD55 expression in vitro. To prepare multi‐transgene “packages” for introduction into animals, the most effective constructs were combined with one or two further xenoprotective transgenes, including A20 (4), HO‐1 (5), thrombomodulin (6) and CTLA4‐Ig (LEA 29Y). In cotransfection experiments single cell clones expressing up to five different xenogenes could be detected. Achieving desired levels of transgene expression in an animal is not only a function of the transgene construct, but also of the location at which it integrates. Position effect variation in expression of random transgenes is well‐documented. Transgene placement by gene targeting at a permissive locus offers a useful means of achieving reliable transgene expression. In mice the ROSA26 locus is widely used to support ubiquitous expression of inserted transgenes (7–8). Murine ROSA26 encodes no functional genes and extensive gene targeting of this locus has not led to deleterious effects on development or physiology (9). To enable a similar approach in pigs, we recently identified a strong candidate for the porcine ROSA26 locus. Gene targeted placement of a neomycin reporter gene construct under the control of the ROSA26 promoter showed expression in all examined porcine tissues of all three germ layers. This strongly suggests that porcine ROSA 26 may resemble the murine locus as a suitably permissive site for transgene expression. We are currently proceeding with gene targeting to place various xenoprotective transgene constructs at this locus. References: 1. Dai Y, Vaught TD, Boone J et al. Targeted disruption of the alpha1,3‐ galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 2. Lai L, Kolber‐Simonds D, Park K et al. Production of alpha‐1,3‐galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 3. Miyagawa S, Fukuta D, Kitano E et al. Effect of tandem forms of DAF(CD55) on complement‐mediated xenogeneic cell lysis. Xenotransplantation 20...

show abstract

“…Attempts to transfer MACs from one species to another have been successful in some cases and have failed in others: human MACs (HACs) have been transferred into mouse and Chinese hamster (Guiducci et al 1999;Shinohara et al 2000). A HAC has been recently transferred from a hamster cell line to a mouse embryonic stem cell line (Paulis et al 2007).…”

Section: Introductionmentioning

confidence: 99%

Interspecific transfer of mammalian artificial chromosomes between farm animals

2009

View full text Add to dashboard Cite

It is often desirable to transfer a mammalian artificial chromosome (MAC) from the cells of one species to those of another. Attempts to carry out such transfer have been successful in some cases and have failed in others. In this study we have tested the hypothesis that centromeric DNA sequence similarity could be a useful criterion for determining MAC host range. Homology studies indicated that the sheep should give positive transfer results. The prediction was tested by introducing into sheep cells a yeast artificial chromosome that contained swine centromeric sequences and that had previously been used to produce a de novo MAC in swine cells. The experiments resulted in the formation of a functional de novo MAC in sheep cells, as attested by FISH analysis. The newly formed MAC remained structurally and functionally stable in ovine up to 52 generations. The centromeric sequences present on the newly formed MAC are probably swine sequences, although it cannot be ruled out that some sheep sequences may also have migrated to the MAC. The size of the sheep MAC was determined by atomic force microscopy. Thus, centromeric sequence similarity appears to be a useful criterion for predicting the animal species between which MACs can shuttle.

show abstract

Use of a Human Minichromosome as a Cloning and Expression Vector for Mammalian Cells

Cited by 28 publications

References 36 publications

Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Efficient Male and Female Germline Transmission of a Human Chromosomal Vector in Mice

Multi‐transgenic pigs for xenotransplantation

Interspecific transfer of mammalian artificial chromosomes between farm animals

Contact Info

Product

Resources

About