Suitability of a CMV/EGFP cassette to monitor stable expression from human artificial chromosomes but not transient transfer in the cells forming viable clones

Abstract

Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1–8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4–6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation.

Bacterial transfer of large functional genomic DNA into human cells

“…The NotI insert (116 kb) of PAC E1 was cloned into the Bsp120I site of pTT (26 kb), a tetratelomeric PAC vector derived from pTAT-BS by insertional duplication of the portion between the telomeres, insertion of a prokaryotic white/blue selectable marker derived from pUC19 (Gibco BRL, Gaithersburg, MD, USA) and insertion of a eukaryotic CMV/EGFP expression cassette derived by PCR from plasmid pEGFP-N1 (Clontech, Palo Alto, CA, USA). 22 Construction of the genomic CFTR expression plasmid CGT21 (EMBL/Genbank accession number BN000167) is described elsewhere. 23 In brief, an unaltered PAC containing the 5 0 portion of the human CFTR locus (À60 kb to CFTR intron 9) was joined by cloning with a synthetic exon fused from sequences of CFTR intron 9, exon 10, the EGFP coding region, CFTR exon 24 plus 3 0 UTR and further CFTR 3 0 downstream region.…”

“…Human artificial chromosomes (HACs) based on alpha satellite DNA as the only human component in a circular PAC or on linear, telomerized alpha satellite DNA, faithfully replicate and segregate during mitosis for many cell divisions even in the absence of selection. 5 Specific alpha satellite DNA arrays from various human chromosomes, such as chromosomes 14,17,21,22, X (and Y inefficiently), have been transferred as purified DNA in order to form centromeres on de novo HACS. [5][6][7][8][9][10][11][12][13] The centromeres formed on the transferred DNA acquire chromatin proteins specific for functional centromeres such as CENP-A a histone H3 variant, only found within active centromere chromatin.…”

“…These vectors included two PACs containing alphoid DNA, TTE1 22 and B2T8, of 150 and 200 kb, respectively, which could form HACs upon cell transfer, and an engineered 160 kb PAC construct, CGT21, which contains a large portion of the human locus containing the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene fused to the EGFP gene as a marker. 23 We demonstrate, for the first time, that bacterial gene delivery systems allow stable clone formation after transfer into cells of low copy number genetic elements of large size, either through the formation of free episomal HACs or through their integration into the host chromosome.…”

Multi‐transgenic pigs for xenotransplantation

“…The tetra-telomerized construct was thought to stabilize the chromosomal ends via T-loops and to result in a higher amount of HAC formation. However, the frequency of HAC formations using the pTTE1 construct was comparable to the di-telomerized constructs as shown by Laner (2004). For the delivery of the xeno-HAC components into the cell lipofection, microinjection and bactofection are possible methods.…”

“…Previous experiments at the chair to achieve HAC formation in porcine cells had failed. All cotransfections of various combinations of linearized xeno-constructs (CD55-XS, CD55-TM-Lea, CD55-HO-A20, GAG-CD55-XS, CAG-CD55-XS-TM-Lea, CAG-CD55-S-HO-A20, CD46-M, CD59-M) were performed in HT1080 cells together with 10-200 ng NotI-digested, tetra-telomerised PAC construct (pTTE1) containing 116 kb of human chromosome 5 alpha satellite DNA, a CMV/EGFP expression cassette and two BS resistance genes (Laner et al, 2004). The pTTE1 plasmid was digested in the plug, separated via PFGE, excised and subsequently electroeluted.…”