Construction of the targeting vector
The `arms' of the targeting vector homologous to brachyury sequences were isolated from a 129/Ola strain BAC library (Genome Systems). The final targeting construct consisted of: (1) a `short arm' of homology encompassing nucleotides -1243 to +3 of the brachyury gene (the adenine in the ATG start codon counting 1); (2) a GFP mini gene as described in the `Results' section; (3) a loxP flanked neomycin resistance gene (Gu et al., 1993); (4) a `long arm' of homology encompassing nucleotides +364 to +7515 of the brachyury gene; and (5) the HSV thymidine kinase gene for negative selection. A detailed description of all cloning steps giving rise to the final targeting vector can be obtained directly from H.J.F. ([email protected]
Generation of brachyury/EGFP knock-in ES cells
Mouse embryonic stem cells (E14.1, 129/Ola) were electroporated with the NotI-linearized targeting vector. Clones that had undergone a homologous recombination event were identified by PCR with one primer (5'-CAGGTAGAACCCACAACTCCGAC-3') annealing to genomic sequences in the 5' region of the brachyury gene, upstream of the `short arm of homology', the other primer (5'-CCGGACACGCTGAACTTGTGGC-3') to the 5' region of EGFP. Correctly targeted clones were confirmed by Southern blot analysis. Out of 384 singly selected and 80 doubly selected colonies, four and three correctly targeted clones were identified, respectively. Two positive clones (#164 and #201) were transiently transfected with a modified Cre recombinase expression vector (H.J.F., unpublished) to excise the neo gene. Neo-deficient clones were identified due to loss of G418 resistance. The intactness of the targeted locus before and after Cre-mediated excision of Neo was confirmed by Southern blot analysis. The absence of the neo cassette in Cre-treated G418-sensitive clones was verified by Southern blotting using the Neo cassette as probe (not shown).
ES cell growth and differentiation
ES cells were maintained on irradiated embryonic feeder cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum (FCS), penicillin, streptomycin, LIF (1% conditioned medium) and 1.5x10-4 M monothioglycerol (MTG; Sigma). Two days prior to the onset of differentiation, cells were transferred on gelatinized plates in the same media. For the generation of EBs, ES cells were trypsinized and plated at various densities in differentiation cultures. Differentiation of EBs was carried out in 60 mm petri grade dishes in IMDM supplemented with 15% FCS, 2 mM L-glutamine (Gibco/BRL), transferrin (200 µg/ml), 0.5 mM ascorbic acid (Sigma), and 4.5x10-4 M MTG. For reaggregation, sorted cells were cultured in the media used to differentiate EBs. Cells were cultured for 20 hours at a density of 4x105/ml in ultra low attachment 24-well plates (Costar). Cultures were maintained in a humidified chamber in a 5% CO2/air mixture at 37°C.
For the generation of blast cell colonies (BL-CFC assay), EB cells were plated in 1% methylcellulose supplemented with 10% FCS (Summit), vascular endothelial growth factor (VEGF; 5 ng/ml), Kit ligand (KL; 1% conditioned medium), IL6 (5 ng/ml) and 25% D4T endothelial cell conditioned medium (Kennedy et al., 1997). For the growth of hematopoietic precursors, cells were plated in 1% methylcellulose containing 10% plasma-derived serum (PDS; Antech), 5% protein-free hybridoma medium (PFHM-II; Gibco-BRL) and the following cytokines: KL (1% conditioned medium), TPO (5 ng/ml), erythropoietin (2 U/ml), IL11 (25 ng/ml), IL3 (1% conditioned medium), GM-CSF (3 ng/ml), G-CSF (30 ng/ml), M-CSF (5 ng/ml) and IL6 (5 ng/ml). Cultures were maintained at 37°C, 5% CO2. LIF and Kit ligand were derived from media conditioned by CHO cells transfected with LIF and KL expression vectors, respectively (kindly provided by Genetics Institute). IL3 was obtained from medium conditioned by X63 AG8-653 myeloma cells transfected with a vector expressing IL3 (Karasuyama and Melchers, 1988). VEGF, GM-CSF, M-CSF, G-CSF, TPO, IL6 and IL11 were purchased from R&D Systems.
Both GFP positive and negative populations were isolated from day 2.5 EBs by cell sorting. Pre-sort and sorted cells were reaggregated at 105 cells/ml in ultra low attachment 24-well plates (Costar) in IMDM supplemented with 15% serum replacement media (Gibco BRL). Twenty-four hours later, the reaggregated EB-like structures were moved to 60 mm petri dishes in the same medium and cultured for an additional 3.5 days. At this stage, the EBs were harvested and transferred to gelatin-coated dishes for the evaluation of neurite formation or to gelatin-coated cover slip for specific staining. Four days later, the proportion of EBs that generated neurites was scored. For immunohistochemical staining, EBs were fixed in 2% paraformaldehyde for 20 minutes, washed twice in PBS, permeabilized in 0.2% Triton X-100/PBS, washed in 10% FCS, 0.2% Tween 20/PBS, and then blocked with 10% FCS/PBS for 10 minutes. EBs were incubated for 1 hour with an antibody against the neuronal class III ß-tubulin (TuJ1; Babco). Bound antibodies were visualized using a secondary Cy3-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories).
Gene expression analysis
The preparation and analysis of 3' UTR cDNA was performed as previously described (Robertson et al., 2000). For gene-specific PCR, total RNA was extracted from each sample with an RNeasy mini kit and treated with Rnase-free DNase (Qiagen). Two micrograms of total RNA was reverse-transcribed into cDNA with random hexamer using an Omniscript RT kit (Qiagen). The PCR reactions were performed with 2.5 U of Taq polymerase (Promega), PCR buffer, 2.5 mM MgCl2, 0.2 µM of each primers and 0.2 mM dNTP. Cycling conditions were as follows: 94°C for 5 minutes followed by 35 cycles of amplification (94°C denaturation for 1 minute, 60°C annealing for 1 minute, 72°C elongation for 1 minute) with a final incubation at 72°C for 7 minutes. PCR was carried out using the following gene specific oligonucleotides: ß-actin, 5'ATG AAG ATC CTG ACC GAG CG3' (sense) and 5'TAC TTG CGC TCA GGA GGA GC3' (antisense); brachyury, 5'CAT GTA CTC TTT CTT GCT GG3' (sense) 5'GGT CTC GGG AAA GCA GTG GC3' (antisense); Runx1, 5'CCA GCA AGC TGA GGA GCG GG3' (sense) 5'CGG ATT TGT AAA GAC GGT GA3' (antisense); Flk1, 5'CAC CTG GCA CTC TCC ACC TTC3' (sense) 5'GAT TTC ATC CCA CTA CCG AAA G3' (antisense); Nodal, 5'CCG TCC CCT CTG GCG TAC ATG3' (sense) 5'GAC CTG AGA AGG AAT GAC GG3' (antisense); Pax6, 5'GCT TCA TCC GAG TCT TCT CCG TTA G3' (sense) 5'CCA TCT TTG CTT GGG AAA TCC G3' (antisense); Rex1, 5'CGT GTA ACA TAC ACC ATC CG3' (sense) 5'GAA ATC CTC TTC CAG AAT GG3' (antisense); Fgf5, 5'AAA GTC AAT GGC TCC CAC GAA3' (sense) 5'CTT CAG TCT GTA CTT CAC TGG3' (antisense); Bmp2, 5'GAA TCA GAA CAC AAG TCA GT3' (sense) 5'GTT TGT GTT TGG CTT GAC GC3' (antisense); Bmp4, 5'TGT GAG GAG TTT CCA TCA CG3' (sense) 5'CAG CGA AGG ACT GCA GGG CT3' (antisense); Wnt3a, 5'GGA ATG GTC TCT CGG GAG TTT G3' (sense) 5'AGG TTC GCA GAA GTT GGG TGA G3' (antisense); Wnt8a, 5'CTG CCT GGT CAG TGA ACA ACT TC3' (sense) 5'GAG TCT GGA GAT TTT TTC CCC G3' (antisense).
Flow cytometry and cell sorting
EBs were harvested, trypsinized and the single cell suspension analyzed on a Facscalibur flow cytometer (Becton Dickinson) or sorted on a Moflo cell sorter (Cytomation Systems). Staining with mAb Flk1 bio, Kit-PE or CD31-bio (PharMingen) was performed as previously described (Kouskoff et al., 2000).