Construction of neocentromere-based human minichromosomes by telomere-associated chromosomal truncation Richard Saffery*, Lee H. Wong*, Danielle V. Irvine, Melissa A. Bateman, Belinda Griffiths, Suzanne M. Cutts, Michael R. Cancilla, Angela C. Cendron, Angela J. Stafford, and K. H. Andy Choo† The Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road, Melbourne 3052, Australia Edited by John A. Carbon, University of California, Santa Barbara, CA, and approved March 1, 2001 (received for review October 3, 2000) Neocentromeres (NCs) are fully functional centromeres that arise chromosomes. We describe here the production of mitotically ectopically in noncentromeric regions lacking ␣-satellite DNA. stable NC-based human MiCs containing a fully functional Using telomere-associated chromosome truncation, we have pro- human NC derived from the 10q25 region of the mardel(10) duced a series of minichromosomes (MiCs) from a mardel(10) marker chromosome (27, 28). marker chromosome containing a previously characterized human NC. These MiCs range in size from Ϸ0.7 to 1.8 Mb and contain Experimental Protocols single-copy intact genomic DNA from the 10q25 region. Two of Cell Culture and Transfection. BE2Cl-18–5f (abbreviated 5f) was these NC-based Mi-Cs (NC-MiCs) appear circular whereas one is cultured as described (28). HT1080 and derivatives were cultured in linear. All demonstrate stability in both structure and mitotic DMEM (GIBCO͞BRL) with 10% FCS. Hygromycin (Roche Mo- transmission in the absence of drug selection. Presence of a lecular Biochemicals), puromycin (Sigma), or zeocin (Invitrogen) functional NC is shown by binding a host of key centromere- were added to medium at 250 ␮g͞ml, 1 ␮g͞ml, or 200 ␮g͞ml, associated proteins. These NC-MiCs provide direct evidence for respectively. Microtubule-depolymerizing agents colcemid mitotic segregation function of the NC DNA and represent exam- (GIBCO͞BRL) or nocadazole (Sigma) were added at 10 ␮Mor0.1 GENETICS ples of stable mammalian MiCs lacking centromeric repeats. ␮g͞ml for 1 or 6–12 h, respectively, before cell harvesting. Transfection of 5f and ZB30 cells was performed by electro- ammalian artificial chromosomes have several potential poration (1.2 kV, 25 uF; Bio-Rad Gene Pulser Electroporator). Mbiotechnological and therapeutic applications arising from Transfection of HT1080 or derivatives was performed by elec- their ability to exist episomally, carry large DNA inserts, and troporation (29) or lipofection. For lipofection, cells were plated allow expression of genes independently of the host genome. By 1 day before transfection to give 60–70% confluency at time of transfection. Two milliliters of diluted Fugene 6 transfection analogy with their yeast counterparts, it has been assumed that ␮ ␮ mammalian artificial chromosomes require a functional mam- reagent (100 l in a total of 2 ml containing 20 g DNA) (Roche malian centromere, telomeres, and DNA replication origins for Molecular Biochemicals) was added onto cells drop-wise, and proper segregation. At present, the least understood and most selection was applied 24–48 h posttransfection. complex of these three components is the centromere. The identification of many protein components necessary for Microcell-Mediated Chromosome Transfer. Microcell fusion was car- correct centromere activity, and the characterization of centromere ried out as described (30). Log-phase donor ZB30 cells arrested in colcemid for 48 h were resuspended in percoll͞serum-free DMEM DNA sequences in a variety of species, have greatly increased our ␮ ͞ knowledge of the mechanisms underlying centromere formation (1:1) supplemented with 20 g ml of cytochalasin B (Sigma). The and function (1–3). This knowledge has facilitated the development cell suspension was subjected to centrifugation at 18,000 rpm, 90 of a number of strategies for mammalian artificial chromosome min, 32°C. Both bands of cell-mix were pelleted, washed with construction. One strategy involves the de novo formation of serum-free DMEM, and filtered through isopore membranes of 30, 8, and 5 ␮M (Millipore). Microcells were resuspended in serum- artificial chromosomes by transfection of large arrays of human ␮ ͞ ␣-satellite into human cells (4–7). Although some of the generated free DMEM containing 10 g ml phytohemagglutinin-P (Difco), artificial chromosomes were linear in structure (4), others were agglutinated with recipient HT1080 cells for 45 min at 37°C, and fused by addition of 50% polyethylene glycol (Roche Molecular consistently circular (5, 7, 8) and all were typically 1 or more orders Biochemicals) for 2 min at room temperature. Cells then were of magnitude larger than the input DNA. rinsed and cultured overnight in DMEM containing 10% FCS A second strategy involves the use of telomere-associated chro- followed by addition of selection. mosome truncation to remove nonessential chromosome arms to produce minichromosomes (MiCs) in situ. Sequential truncation of Fluorescence in Situ Hybridization (FISH), Immunofluorescence, and a human X and Y chromosome has yielded a number of ␣-satellite- Pulsed-Field Gel Electrophoresis (PFGE). Combined FISH͞ containing MiCs of varying stability and sizes ranging from Ϸ0.7 immunofluorescence was performed as described (25). FISH Mb to more than 4 Mb (9–15). A final strategy for production of using pan-␣-satellite probe pTRA7 and PNA-FISH of telomeric mammalian artificial chromosomes involves the amplification of pericentric DNA followed by controlled breakage of chromosomes to produce satellite DNA-based artificial chromosomes of between This paper was submitted directly (Track II) to the PNAS office. 60 and 400 Mb (16, 17). These artificial chromosomes express Abbreviations: NC, neocentromere; MiC, minichromosome; FISH, fluorescence in situ hy- exogenous genes and can be stably introduced into different bridization; PFGE, pulse-field gel electrophoresis; DAPI, 4Ј,6-diamidino-2-phenylindole; mammalian cell lines and transgenic mice (18–20). BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome. Neocentromeres (NCs) lacking the repeat sequences tradi- See commentary on page 5374. tionally associated with centromere function recently have been *R.S. and L.H.W. contributed equally to this work. described (21, 22). Characterization of NCs in humans suggests †To whom reprint requests should be addressed. E-mail: [email protected]. epigenetic mechanism of formation independent of primary The publication costs of this article were defrayed in part by page charge payment. This DNA sequence composition (23–26). The discovery of NCs article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. provides an alternative approach to the construction of artificial §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.091468498 PNAS ͉ May 8, 2001 ͉ vol. 98 ͉ no. 10 ͉ 5705–5710 Downloaded by guest on September 25, 2021 sequences (PerSeptive Biosystems, Framingham, MA) were DNA into the host genome by FISH, screening of more than 450 performed as described (31, 32). Chromosome painting was drug-resistant cell lines failed to identify any containing an performed by using a WCP Chromosome Paint Kit (Vysis). activated NC either in the form of a stable MiC or as the result Subchromosome-10 DNA paints were derived from somatic cell of insertion of the introduced DNA into HT1080 genome (data radiation hybrid obtained from M. Rocchi (University of Bari, not shown). In an alternative approach, a 640-kb yeast artificial Bari, Italy). InterAlu amplification of somatic cell hybrid DNA chromosome (YAC) clone (YAC-3) (28) containing the core NC was carried out by using primers 5Ј-GGATTACAGGYRT- region was retrofitted with human telomeric DNA and selectable GAGCCA and 5Ј-RCCAYTGCACTCCAGCCTG as de- markers by using the plasmid vectors pRANT 11 and pLGTEL scribed (33). 1, following published procedures (46). Repeated transfection of Polyclonal anti-CENP-A, monoclonal anti-CENP-B, poly- correctly retrofitted YACs into human and Chinese hamster clonal anti-CENP-C, and CREST6 antisera were as described ovary cells using spheroplast fusion resulted in YAC DNA (28, 34, 35, 36). Polyclonal anti-CENP-E (37), anti-CENP-F (38), uptake, insertion into the Chinese hamster ovary genome, YAC and anti-hBUB1 (39) were kindly provided by T. Yen (Fox Chase DNA amplification, and double minute formation, but no acti- Cancer Center, Philadelphia), polyclonal anti-hZW10 (40) by B. vation of NC or artificial chromosome formation (data not Williams and M. Goldberg (Cornell University, Ithaca, NY), shown). Despite extensive efforts, our inability to recover func- polyclonal p55CDC (41) by J. Weinstein (Amgen Biologicals), tional artificial chromosomes by using in vitro assembly ap- and polyclonal anti-TRF1 (42) by T. deLange (The Rockefeller proaches prompted us to focus on the use of a different strategy University, New York). involving in situ chromosome truncation to generate MiCs. High molecular weight genomic DNA was prepared (43) and run on pulsed-field gels (Bio-Rad CHEF Mapper System) in Mardel(10) Tagging and Transfer into HT1080 Cells. We previously 0.5 ϫ Tris-acetate͞EDTA with buffer changes every 2 days. have created a somatic cell hybrid line (designated 5f) containing mardel(10) (28) in a Chinese hamster ovary background. Telomere- -Size Determination by 4؅,6-Diamidino-2-phenylindole (DAPI) Staining. associated chromosome truncation (TACT) (9, 12, 44) was per Metaphase spreads were stained in 600