Transfer of a Human Chromosomal Vector from a Hamster Cell Line to a Mouse Embryonic Stem Cell Line

Paulis, Marianna; Bensi, Mirella; Orioli, Donata; Mondello, Chiara; Mazzini, Giuliano; D’Incalci, Maurizio; Falcioni, Cristiano; Radælli, Enrico; Erba, Eugenio; Raimondi, Elena; Carli, L. De

doi:10.1634/stemcells.2007-0052

Cited by 15 publications

(10 citation statements)

References 25 publications

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“…The labelled probes were re-suspended in hybridization buffer (50% formamide, 10% dextran sulphate, 1x Denhart's solution, 0.1% SDS, 40 mM Na 2 HPO 4 pH 6.8, 2xSSC) containing 10X mouse Cot1 DNA (Life Technologies) and denatured at 80°C for 10 min. In situ hybridization was performed as previously described [ 25 ]. In brief, slides were treated with Pepsin (0.004%) at 37°C for 30 sec and dehydrated through the ethanol series before denaturation in 70% formamide/2xSSC.…”

Section: Methodsmentioning

confidence: 99%

Chromosome transplantation as a novel approach for correcting complex genomic disorders

et al. 2015

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Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt gene with a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

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Section: Methodsmentioning

confidence: 99%

Chromosome transplantation as a novel approach for correcting complex genomic disorders

et al. 2015

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“…FISH experiments were carried out on interphase and metaphase cells from transfected ES cells as previously described 26 . Briefly, cell cultures were treated with KaryoMAX colcemid (Life Technologies) at a final concentration of 0.1 μg/ml for 2 h at 37 °C and cells were then detached by treatment with 0.25% trypsin/ EDTA (Lonza).…”

Section: Methodsmentioning

confidence: 99%

A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

Paulis

Castelli

Lizier

et al. 2015

Sci Rep

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The clustered regularly interspaced short palindromic repeat (CRISPR)/associated 9 (Cas9) technology has been recently added to the tools allowing efficient and easy DNA targeting, representing a very promising approach to gene engineering. Using the CRISPR/Cas9 system we have driven the integration of exogenous DNA sequences to the X-linked Hprt gene of mouse embryonic stem cells. We show here that a simple fluorescence in situ hybridization (FISH)-based strategy allows the detection and the frequency evaluation of non-specific integrations of a given plasmid. FISH analysis revealed that these integrations do not match the software predicted off-target loci. We conclude that the frequency of these CRISPR-mediated off-target DNA cuts is negligible, since, due to the occurrence of spontaneous double-strand breaks, we observed more aspecific plasmid integrations than those corresponding to predicted off-target sites.

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“…The transfer of a HAC from a hamster cell line to a mouse ES cell line was reported by Paulis et al, (2007). However, attempts to transfer a HAC into porcine cells or a porcine minichromosome into human cells were so far not successful (Cavaliere et al, 2009).…”

Section: Human Artificial Chromosomes and Microcell Mediated Chromosomentioning

confidence: 99%

Multi‐transgenic pigs for xenotransplantation

Fischer

Kraner-Scheiber

Flisikowski

et al. 2013

Xenotransplantation

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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...

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Transfer of a Human Chromosomal Vector from a Hamster Cell Line to a Mouse Embryonic Stem Cell Line

Cited by 15 publications

References 25 publications

Chromosome transplantation as a novel approach for correcting complex genomic disorders

Chromosome transplantation as a novel approach for correcting complex genomic disorders

A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

Multi‐transgenic pigs for xenotransplantation

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