Functional Complementation of a Genetic Deficiency with Human Artificial Chromosomes

Abstract

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.

Comparative study of artificial chromosome centromeres in human and murine cells

“…Similar to the analysis of HAC in previous work, 8 HAC 1-H and its murine derivative HAC 1-M1 contained 17a DNA interspersed with HPRT sequences. The FISH signals were of variable length, confirming that the input DNA had undergone some amplification and rearrangement ( Figure 1b).…”

“…In previous work, the AG6-1 HAC was obtained in HT1080 cells, following lipofection delivery of an input vector containing 220 kb of 17 alpha DNA and the whole HPRT genomic locus (200 kb). 8 The AG6-1 HAC was transferred by MMCT to STO murine cells, to generate Sag1.2 (Moralli et al 9 ). The LJ2-1 HAC was obtained in HT1080 from a vector containing 220 kb of 17a DNA on a pBeloBAC11 backbone.…”

Visible transient expression of EGFP requires intranuclear injection of large copy numbers

“…Human artificial chromosomes (HACs) are presently constructed to understand how chromosomes work and which sequence elements are required for stability and function. [1][2][3][4][5][6] Ultimately, HACs could become a safe and efficient gene transfer tool circumventing DNA-induced cell cycle arrest, replicative loss, integrative repair, and silencing. 7,8 In a series of HAC transfection experiments we recently established that the inclusion of large genomic DNA (100 kb) capped by telomeric repeats improves the de novo formation of HACs based on cloned centromere DNA, making HAC formation more efficient than the conventional procedure to obtain stable clones via integration into host chromosomes (Schindelhauer et al, in preparation).…”

Chromosome engineering: prospects for gene therapy

“…7,10,31,32 The recent demonstration that circular HACs can be formed from circular PAC DNA lacking telomere repeats and containing 70 kb of alpha satellite DNA, represents a step forward in determining the minimal requirements for generating a mitotically stable HAC. 7 Previous vectors included telomere repeats, the aim being to generate a linear HAC reflecting host chromosome structure.…”

“…In one approach, a BAC-based vector was constructed that was linearized to generate a 404 kb telomere capped construct containing 220 kb of alpha satellite DNA and a 160 kb fragment containing a human HPRT gene (Figure 2a). 32 In the second approach, a co-transfection strategy was used combining a PAC containing 70 kb of alpha satellite DNA with a second (160 kb) genomic PAC spanning a human HPRT gene (Figure 2b). 33 Lines were developed containing an HPRT-expressing HAC as the sole fate of the transfected DNA (Figure 2c).…”