Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays

Abstract

Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.

Figure 1

(A) Outline of iterative scheme for synthesis of mutated versions of D17Z1 alpha-satellite arrays. Each of the 16 individual monomers comprising a single 2.7 kb higher-order repeat (HOR) was synthesized as 2–3 oligonucleotide pairs (60–100 bp each), which were directly ligated together and gel purified. Adjacent HORs were subsequently ligated to form dimers as shown, and PCR modified to introduce SapI recognition sites at both ends as appropriate. Digestion with SapI allows seamless ligation of adjacent dimers to create tetramers without introduction of extraneous non-alpha-satellite sequences. Two additional rounds of serial ligation resulted in formation of a complete synthetic HOR, which was subcloned into the BAC vector pBeloBAC. (B) Outline of scheme for directional multimerization of mutated HORs. A synthetic, 86 kb alpha-satellite array consisting of 32 tandem copies of the HOR was created as follows. pBAC17α1 was digested with BglII and SpeI and the alpha-satellite containing fragment (fragment ‘A’) was isolated and gel purified. The same construct was separately digested with BamHI and SpeI, and the larger fragment (fragment ‘B’) was isolated and gel purified. Ligation of fragment ‘A’ to fragment ‘B’ is directional, resulting in head-to-tail multimerization of adjacent repeats. The resulting pBAC17α(n + 1) construct was then isolated following transformation of the ligation reaction into E.coli. This process was repeated iteratively to create the final pBAC17α32 arrays. (C) Pulsed-field gel electrophoresis (PFGE) analysis of intermediates in the construction of 17α32 HOR/BeloBAC constructs. Each intermediate was digested with NotI, which drops out the entire subcloned alpha-satellite array from the pBeloBAC vector backbone. Each lane shows the completed intermediate en route to 32 copies of the mutated HOR. The number beneath each lane corresponds to the number of copies of the HOR in each construct, and the arrows to the right indicate the size of the alpha-satellite insert and the vector backbone. The insert in lane labeled ‘1’ is 2.7 kb and therefore too small to be resolved by PFGE.

Figure 2

Cytogenetic analysis of HACs from synthetic chromosome 17-derived alpha-satellite arrays. Arrows designate HACs. Immunostaining with an anti-CENP-C antibody (green) identifies functional centromeres. FISH with the synthetic alpha-satellite as probe (red) hybridizes with the HAC as well as to the centromeres of the endogenous HT1080 chromosome 17s. (HT1080 is quasitetraploid, so there are four copies of the endogenous chromosome 17s.) DAPI-stained chromosomes are shown in blue. (A) HT1080 clone generated by transfection with pBAC17α32-all construct (Table 1) showing the presence of two HACs. (B) HT1080 clone generated by transfection with pBAC17α32-natural. A single HAC is visible. (C) HT1080 clone generated by transfection with pBAC17α32-null. Two HACs are present.

Figure 3

Digestion of pBAC 17α32 HTH Tel TN with PshA1 releases the ∼100 kb linear transposon vector, shown with an 86 kb chromosome 17-derived alpha-satellite array, as well as ∼800 bp synthetic telomeres separated by a recognition site for the ultrarare endonuclease I-CeuI. Mammalian cell line selection is based on a pgk-puro cassette conferring resistance to puromycin and a neo/kan cassette conferring resistance to geneticin. The transposon vector is flanked by 19 bp transposase recognition elements (ME).

Figure 4

Detection and validation of transposition events in genomic BACs. (A) Identification of transposition events by NotI digest. Lane 1, target genomic BAC. Lane 2, transposon insertion into genomic region of target BAC. Note that insert fragment excised by NotI is much larger than the starting fragment from lane 1. Lane 3, transposon insertion into vector backbone of target BAC. Note that vector fragment is much larger than the starting vector fragment from lane 1, while genomic insert fragment is unchanged. (B) Confirmation of successful transposition of complete 86 kb alpha-satellite array. Lane 1, target BAC cut with NotI. Lane 2, target BAC cut with BamHI. Lanes 3–5, clone with putative transposition event cut with I-CeuI (lane 3), Not I (lane 4) or BamHI (lane 5). The 86 kb band in lane 5 represents the size of the alpha-satellite array present in the clone. (C) Analysis of alpha-satellite array integrity in BAC modified by transposition. pBAC17α32 HTH Tel TN control cut with PstI (lane 1), PvuII (lane 2), EcoRI (lane 3) and HindIII (lane 4). Clone with putative transposition event cut with PstI (lane 5), PvuII (lane 6), EcoRI (lane 7) and HindIII (lane 8). Both PstI and PvuII digests generate a multicopy 2.7 kb HOR fragment from the alpha-satellite array. HindIII is predicted to produce bands at 1.4 and 0.9 kb. In A, B and C, L and H represent low-molecular weight and high-molecular weight markers, respectively.

Figure 5

Cytogenetic analysis of HACs created from unimolecular BAC–HAC vectors. (A) Dual FISH/immunostaining with anti-CENP-C antibodies (red) and D17Z1 probe (green). (B) Two-color FISH analysis: D17Z1 probe (green) and 150 kb genomic fragment probe from BAC–HAC vector (red). (C) Two-color FISH analysis: D17Z1 probe (green) and BAC vector backbone probe (red). (D) Two-color FISH analysis: D17Z1 probe (green) and telomeric DNA (red). In all panels, DAPI-stained DNA in blue.