Transposon-mediated BAC transgenesis in human ES cells

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.


Supplementary Information.
. Diagrams of the reporter constructs for OCT4 and NANOG genes.       Recombineering efficiency shown as colonies obtained (CFUs) after ampicillin selection for the insertion of the PiggyBac ITR cassette into pBACe3.6. The ITR cassette was prepared in four different ways. Either it was (i) cut with both PacI and AscI, then the 5' ends were dephosphorylated (PacI/AscI)-dP; (ii) cut first with PacI, dephosphorylated, then cut with AscI (PacI-dP)/AscI; (iii) cut first with AscI, dephosphorylated, then cut with PacI (AscI-dP)/PacI; or (iv) double digested with PacI/AscI. The best efficiency was observed when the vector was digested first with AscI, dephosphorylated and then digested with PacI because the dephosphorylated strand primes Okazaki-fragment synthesis and is more recombinogenic.
See (Maresca et al., 2010) for a fuller explanation. (e) Recombineering strategy for placing the inverted repeats at either ends of BAC backbone (9,5kb apart).    (73). However, with a circular substrate like a BAC, the distance between the ITRs remains small, even though the transposon can exceed 100kb. The model postulates that ITR proximity but not orientation is critical and that transposition is indifferent to cargo size. It also implies that the efficiency of large transposition depends on a covalently closed circular substrate.
The large constructs were made by subcloning from the respective BACs a region of 19 kb for hOCT4 gene and 25 kb for hNANOG into a plasmid with p15A origin of replication using recombineering technology, Figure S1 ( Zhang et al., 1998;Zhang et al., 2000) . In brief, the p15A origin together with the ampicillin resistance gene were amplified by PCR using the primers hOct4-sub1, hOct4-sub2 for the hOCT4 and hNanog-sub1, hNanog-sub2 for the hNANOG region. For the generation of large construct-or BAC-reporters, the GFP or Cherry cassettes were inserted directly after the initiating Methionine (ATG) of the respective gene using recombineering. For the BACs, the recombinase expression plasmid pSC101-BAD-gbaA-tet was electroporated into the BAC host cells and colonies were selected using Chloramphenicol (Cm) and Tetracycline (Tet) at 30°C. The recombination proficient BAC host was electroporated with the PCR-amplified eGFP or mCherry cassettes, which were flanked by the homologous sequences to the gene. Recombined colonies were selected with Kanamycin (Km) and Cm at 37°C. A similar protocol was used to insert the cassettes into the large constructs using ampicillin (Amp) and Tet selection for introducing pSC101-BAD-gbaAtet and Amp and Km selection for the final step. The list of recombineering oligo's is presented below.
Finally a cassette containing the PiggyBac or Sleeping Beauty terminal repeats was inserted into the BAC backbone by recombineering ( Figure S2). The PiggyBac cassette was constructed by replacing the content between the terminal repeats in pPB-CAG-OSKM-puΔtk plasmid with a PCR cassette comprised by the Hygromycin resistance gene, the R6K-origin, restriction sites AscI and PacI for later removal and homology arms to the BAC backbone.
Then the ampicillin resistance gene and ori of the pPB-CAG-OSKM-puDtk vector were replaced by the spectinomycin resistance gene. As a last step the spectinomycin gene was changed again to ampicillin. The Sleeping Beauty cassette was made by first introducing the Hygromycin PCR cassette described above between the SB terminal repeats of the pT2BH vector (provided by Z. I.). Then the vector backbone of pT2BH was exchanged by the spectinomycin resistance gene.
The cassettes were removed from the R6K-vector by different combinations of PacI and AscI digestion and inserted into the BAC backbone. Digestion with AscI first followed by dephosphorylation and digestion with PacI resulted in the highest number of recombined BAC colonies ( Figure S2).
To insert inverted repeats at each end of the BAC backbone we applied sequential recombineering. As a first step an Amp cassette that contained 5'-PB and 5'-SB repeats was inserted upstream of T7 sequence replacing lox511 site. As a second step a cassette

Electroporation of hESCs
Plasmid DNA for electroporation was linearized by restriction digestion, purified by phenolchloroform extraction and dissolved in a small volume of PBS at a concentration of 300 ng/µl. For lipofection hESCs were split to Matrigel-coated dishes in the presence of 10 µM ROCK inhibitor diluting with the ratio 1:3 one day before transfection. 3, 10, 30 or 50 µg of BAC and 3 or 10 µg of the transposase expression or control vector were dissolved in 3 ml of OPTI-MEM medium (Gibco, Invitrogen). Each DNA mix was used for lipofection of a 10 cm dish with hESCs using Lipofectamine LTX (Invitrogen) according to manufacturer protocol. 2 days after transfection 100 µg/ml G418 (Gibco, Invitrogen) or 2 µg/ml blasticidin (Invitrogen) was added to select resistant clones. After 14 days of selection the stable clones were picked to 96-well plates and expanded for freezing and subsequent analysis.

PCR analysis of hESC clones
For PCR analysis the clones of hESCs were expanded in 96-well plates and lysed in 50 µl of buffer containing 0,1 mg/ml proteinase K at 56°C overnight. DNA was precipitated directly on the plate by adding 100 µl of cold ethanol with 75 mM NaCl and incubating for 30 min at room temperature. Supernatant was discarded by carefully inverting the plate and DNA pellet that adhered to bottom of the well was washed with 70% ethanol, dried and dissolved in 50 µl of water. The presence of transposon inverted repeats and loss of ampicillin/spectinomycin cassette in the clones that occurs during transposition, was checked by PCR analysis using the primers listed below. PCR was performed in 50 µl mix containing 1x buffer with 1.  (both from Peprotech). Next day Wnt3a was omitted from the medium, and the cells were cultured further in RPMI with B27 (1x), 0.5mM sodium butyrate and 100 ng/ml activin A with daily medium change. On the day 7 of differentiation the cells were analyzed for GFP and CXCR4 expression by flow cytometry, and for markers expression by qPCR.

Immunostaining and microscopy
Cells on glass cover slips were fixed with 4% formaldehyde in PBS for 10 min at room temperature and washed twice in PBS. The cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked in 0.1% Tween-20 in PBS for 30 min. The incubation with primary antibodies was for 1 hour at room temperature with mouse anti-Oct4 (1:50, sc-5279, Santa Cruz), rabbit anti-Nanog, (1:30, AB5731, Chemicon), or overnight at +4°C with mouse anti-Pax6 (1:30, Developmental Studies Hybridoma Bank). After the wash in PBS, the cells were incubated with secondary antibodies diluted 1:500 (FITC goat anti-rabbit and TRITC goat anti-mouse, Jackson Immunoresearch Laboratories; Alexa633 goat anti-mouse, Molecular Probes, Invitrogen) for 1 hour at room temperature. The nuclear staining was done with DAPI for 3 min, and then the slides were mounted using Mowiol (Fluka). Fluorescence images were taken using Leica SP5 laser scanning confocal microscope.

Flow cytometry.
The cells were dissociated using TrypLE, washed in the culture medium and fixed in PBS with 1% formaldehyde. For antibody staining, 10 6 of live cells were incubated with PE-conjugated