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

Abstract

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.

Chromosome engineering: prospects for gene therapy

“…14,15,17 The production of mice harboring a human chromosome(s) showing correct tissue-specific human immunoglobulin gene expression provides the first mouse model addressing the capacity of a human marker chromosome to deliver tissue specific transgene expression. The Ishida and Marynen laboratories have now taken this technology one step further by modifying human marker chromosomes using the Cre/loxP system to introduce genes that were stably expressed following transfer of the modified marker chromosome into mice.…”

“…The Ishida and Marynen laboratories have now taken this technology one step further by modifying human marker chromosomes using the Cre/loxP system to introduce genes that were stably expressed following transfer of the modified marker chromosome into mice. 16,17 …”

“…The existence of spontaneously arising or radiation-induced human marker chromosomes offers an alternative starting point for deriving a chromosomebased vector system. [11][12][13][14][15][16][17] A further chromosome engineering technology under development relies upon integration of exogenous DNA into the centromere region of a host mouse or human chromosome. Amplification of DNA at the integration site can result in chromosome breakage events, producing new large chromosomes typically in the 60-400 Mb size range.…”

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

“…One approach towards constructing HACs, called "top-down", involves fragmentation of already existing chromosomes and generation of smaller mini-chromosomes, where only the three functional chromosomal elements remain. Several studies have shown that minichromosomes can host and allow the expression of large therapeutic genes, be transferred between various mouse and human cell lines and be transmitted through the mouse germ line (Kakeda et al, 2005;Shen et al, 2001;Voet et al, 2001). Though mini-chromosomes have useful properties for application in transgenics, their use in gene therapy is restricted to an ex vivo approach only.…”

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications

“…Ren et al 2006;Kouprina et al 2013;Kouprina et al 2014). Four different strategies have been developed for the construction of artificial chromosomes (Irvine et al 2005): (i) in the synthetic approach the artificial chromosome is assembled from chromosomal components (Basu et al 2005a;Basu et al 2005b; Harrington et al 1997;Henning et al 1999;Ikeno et al 1998;Ikeno et al 2009;Kaname et al 2005;Kouprina et al 2003;Nakashima et al 2005;Suzuki et al 2006), (ii) the 'top down' method applies the in vivo telomere-associated fragmentation of existing chromosomes (Au et al 1999;Auriche et al 2001;Carine et al 1986;Choo et al 2001;Farr et al 1995;Heller et al 1996;Ishida et al 2000;Katoh et al 2004;Mills et al 1999;Shen et al 2000;Voet et al 2001;Wong et al 2002) , (iii) naturally occurring minichromosomes may be engineered to construct an artificial chromosome (Raimondi 2011), and (iv) de novo chromosome generation can be induced via targeted amplification of specific chromosomal segments (Carl 2004;Csonka et al 2000;Holló et al 1996;Keresö et al 1996;Lindenbaum et al 2004;Praznovszky et al 1991;Tubak et al 1991). To date, two technologies, the 'top down' and the induced de novo chromosome generation approaches have advanced to the point suitable for biotechnological applications.…”