Transfer of HAC-GRY.

<p>(a) HAC-GRY was transferred into NIH3T3 cells by microcell-mediated chromosome transfer and the expression of fluorescent proteins was analyzed by flow cytometry. Top panels, parental NIH3T3 cells; bottom panels, NIH3T3 cell carrying HAC-GRY. For NIH3T3 clones carrying HAC-GRY, the expression of TdTomato and Venus was analyzed in the EGFP⁺ population. (b) HAC (red) and mouse chromosomes (green) were detected by FISH using a digoxigenin-labeled human Cot-1 probe and a biotin-labeled mouse major satellite probe, respectively. Arrows indicate HACs. Enlarged images of HACs are shown in insets.</p

A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

<div><p>Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding <i>EGFP</i>, <i>Venus</i>, and <i>TdTomato</i> into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.</p></div

Combination of simultaneous and sequential integration of GLVs to the HAC.

<p>(a) Schematic representation of simultaneous integration followed by sequential integration of GLVs. (b) PCR analysis of the integration of GLVs to the HAC. (c) The expression of TdTomato was analyzed by flow cytometry. (d) CHO cells carrying the indicated HAC were cultured in medium supplemented with HAT or 800 µg/ml G418, HT, and 10 µg/ml 6TG. Scale bar, 200 µm. (e) HAC (red) and transgenes (green) were detected by FISH using a digoxigenin-labeled human Cot-1 probe and a biotin-labeled <i>TdTomato</i> probe, respectively. Arrows indicate HACs. Enlarged images of HACs are shown in insets.</p

Transfer of MAC1 to HT1080 cells.

<p>(a) MAC1 was introduced into HT1080 cells using PEG-MMCT (PEG) or Am-MMCT (Am). MAC1-transferred G418-resistant HT1080 clones were stained with crystal violet. Representative images of the plates are shown. (b) Comparison of total colony numbers generated by the PEG-MMCT and Am-MMCT methods. Each value represents the mean ± SD (<i>n</i> = 3; *<i>P</i> < 0.01). (c) FISH analysis of a MAC1-transferred HT1080 clone obtained by Am-MMCT. MAC1 (arrow) was detected with a mouse major satellite DNA probe (red). Human chromosomes were detected using a human Cot-1 probe (Green). The sample was counterstained with DAPI to visualize chromosomes (blue). Inset: high magnification image of MAC1. (d) Phase (left panel) or fluorescent (right panel) images of MAC1-transferred HT1080 clone. Scale bar, 100 μm. (e) Genomic PCR using primer sets specific for EGFP expression cassettes of lentivirus vector (EGFP (Lv)) or MAC1 (EGFP (MAC1)). Genomic DNA prepared from indicated cells was used as template. AmCHO-MAC1, AmCHO cells carrying MAC1; HT1080-MAC1, MAC1-transferred HT1080 cells obtained by Am-MMCT. (f) Genomic PCR using a primer set for 3´HPRT cassette of MAC1.</p

Transfer of MAC1 to NIH3T3 cells.

<p>(a) MAC1 was introduced into NIH3T3 cells using PEG-MMCT (PEG), Eco-MMCT (Eco), or Am-MMCT (Am). MAC1-transferred NIH3T3 cells were selected with G418, and colonies were stained with crystal violet. Representative images of the plates are shown. (b) Comparison of total colony numbers generated using the three MMCT methods. (c) FISH analysis of a MAC1-transferred NIH3T3 clone obtained by Eco-MMCT. Chromosomes of mouse origin were detected using a mouse major satellite probe (red). MAC1 (arrow) was identified with a Neo gene probe (green). The sample was counterstained with DAPI to visualize chromosomes (blue). Inset: high magnification image of MAC1. (d) Phase (upper panel) or fluorescent (lower panel) images of MAC1-transferred NIH3T3 clone. Scale bar, 100 μm. (e) Genomic PCR using primer sets specific for EGFP expression cassettes of lentivirus vector (EGFP (Lv)) or MAC1 (EGFP (MAC1)). Genomic DNA prepared from indicated cells was used as template. EcoCHO-MAC1, EcoCHO cells carrying MAC1; NIH3T3-MAC1, MAC1-transferred NIH3T3 cells obtained by Eco-MMCT. (f) Genomic PCR using a primer set for 3´HPRT cassette of MAC1. (g) Efficiency of retro-MMCT using cryopreserved microcells. MAC1 was introduced to NIH3T3 cells using cryopreserved microcells and all three MMCT methods. Each value represents the mean ± SD (<i>n</i> = 3; *<i>P</i> < 0.005; **<i>P</i> < 0.01).</p

Simultaneous integration of GLVs into the HAC.

<p>(a) Schematic representation of the simultaneous integration of GLVs encoding <i>EGFP</i>, <i>TdTomato</i>, and <i>Venus</i> expression units into the HAC. Abbreviations are shown. Arrows indicate the position of PCR primers used for genotyping. (b) PCR analysis of the integration of GLVs into the HAC. Genomic DNA was prepared from HAT resistant CHO clones and CHO cells carrying an empty HAC for the negative control (N). (c) Sequencing analysis of the junctional region of reconstituted <i>HPRT</i>. Predicted sequence is shown in black. HAC-GRY sequence is shown in red. Sequence of each element is underlined. Asterisks indicate matched sequences. (d) GLVs encoding the <i>EGFP</i> expression unit (green box), <i>TdTomato</i> expression unit (red box), <i>Venus</i> expression unit (yellow box), or empty unit (white box) were introduced to CHO cells carrying the HAC. Structure of the GLVs-integrated HAC is depicted on the left. Abbreviations of the generated HACs are shown in the scheme. HAC-E2G, -E2R, and -E2Y were constructed as controls. The expression of fluorescent proteins was analyzed by flow cytometry. Percentages of positive cells in each cell population are shown in the dot plots. For a CHO clone carrying HAC-GRY, expression of TdTomato and Venus was analyzed in the EGFP⁺ population. Three fluorescent proteins were expressed simultaneously in CHO cells carrying HAC-GRY. (e) HAC (red) and transgenes (green) were detected by FISH using a digoxigenin-labeled human Cot-1 probe and biotin-labeled GLVs (GLV1, 2, and 3) probe, respectively. Arrows indicate HACs. Enlarged images of HACs are shown in insets.</p

Sequential integration of GLVs to the HAC.

<p>(a) Schematic representation of the sequential integration of GLVs to the HAC. PCR primers to detect reconstituted selection marker genes are depicted by arrows. (b) Reconstitution of <i>HPRT</i> or <i>5′HPRT-Neo</i> was analyzed by PCR. Note the amplicon from the HAC carrying GLV1, 2, and 3 (HAC-G/R/Y, arrow 1) was slightly larger than that of HAC-G due to attR sequences generated by the second and third round of the integration reaction. (c) The expression of fluorescent proteins was analyzed by flow cytometry. Percentages of positive cells in each population are shown in the dot plots. Expression of TdTomato and Venus was analyzed in the EGFP⁺ population. (d) CHO cells carrying the indicated HAC were cultured in medium supplemented with HAT or 800 µg/ml G418, HT, and 10 µg/ml 6TG. Scale bar, 200 µm. (e) HAC (red) and transgenes (green) were detected by FISH using a digoxigenin-labeled human Cot-1 probe and a biotin-labeled GLVs (GLV1, 2, and 3) probe, respectively. Arrows indicate HACs. Enlarged images of HACs are shown in insets.</p

Efficiency of simultaneous integration of GLVs.

<p>3×10⁵ target cells in a well of 12-well tissue culture plate were transfected with empty GLVs coding the indicated SIM cassettes together with expression vectors for Cre, Bxb1 integrase, and φC31 integrase. Cells were selected with HAT and resistant cells were stained with crystal violet to count the number of colonies.</p><p>Efficiency of simultaneous integration of GLVs.</p

SIM cassettes and GLVs.

<p>(a) Schematic representation of SIM cassettes. SIM cassettes are classified into three groups based on the order of the use. Abbreviations and name of each cassette are shown. (b) A GLV was constructed by inserting a SIM cassette into a gene of interest (GOI)-encoding vector. For the sequential integration, first step is the introduction of a GLV having cassette 1 to the gene-loading site of the HAC using Cre recombinase. The cassette 1 contains Bxb1 attP sequence so that a GLV with cassette 2 can be integrated into the HAC as a second GLV by the use of Bxb1 integrase. φC31 attP sequence of the cassette 2 enables loading of the third GLV with cassette 3 using φC31 integrase. Afterward, alternative use of GVLs having cassette 2 or cassette 3 allows for sequential introduction of GLVs into the HAC. For the simultaneous integration, GLVs having cassette 1, 2 or 3 were loaded to the HAC simultaneously by using Cre, Bxb1 integrase, and φC31 integrase. For the combination of simultaneous and sequential integration of GLVs, cassette 1 containing FRT sequence was used. The FRT sequence makes it possible to integrate a fourth GLV having cassette 1 after the simultaneous integration of three GLVs. Fifth and more GLVs could be introduced in the same way based on the sequential loading procedure.</p

Cell fusion of EnvΔR-expressing CHO cells.

<p>Lentiviral vectors encoding ecotropic (a) or amphotropic (b) EnvΔR-IRES-EGFP were used to infect CHO cells (green, upper panels), which were then co-cultured with CHO, NIH3T3, or 293T cells labeled with TdTomato (red, middle panels). Arrowheads indicate EGFP⁺TdTomato⁺-fused cells. EcoCHO and AmCHO represent CHO cells transduced with ecotropic and amphotropic EnvΔR-IRES-EGFP, respectively. Phase contrast images are shown in the lower panels. Scale bars, 100 μm.</p