Materials and Methods
Generation of Aurka conditional allele
Aurka genomic DNA was isolated from RPCI-22 129S6/SvEvTac Mouse BAC Library,
using Aurora-A cDNA as a probe. From this BAC an 8 kb genomic fragment digested by
XbaI (Fig. 1A) was subcloned and used to generate a conditional Aurora-A targeting
vector. The targeting vector was designed such that exon 2, which contains the ATG start
codon, was flanked with loxP sites. The organization of the targeting vector was as
follows (Fig. 1A): a mc1-TK (thymidine kinase) for negative selection, the left arm
consisting of a 1 kb SalI/PstI fragment contained within intron 1, loxP, Neo (neomycin
resistance) for positive selection flanked by Frt sites, the middle arm consisting of a 1 kb
PstI/BamHI fragment containing exon 2, the second loxP, and the right arm consisting of
a 5.5 kb BamHI/XbaI fragment containing exon 3 through 6. ES cells were targeted as
described (McMahon and Bradley, 1990). The targeting vector was electroporated into
AB1 ES cells. These cells were then plated on mitomycin C treated feeder cells, and
positively selected with G418 and negatively selected with gancyclovir. Identification of
properly targeted ES cell clones and genotyping of mouse tail DNAs were done by
Southern blotting of XbaI/KpnI digests using the 5’ and the 3’ probes. Chimeric mice
from two heterozygous targeted ES cell clones were generated at the M. D. Anderson
Genetically Engineered Mouse Facility.
Mouse breeding
The chimeric mice were crossed to C57B6 mice to produce heterozygous conditional neo
(CN) mice. These mice were crossed to Rosa 26 flipper mice to remove the neomycin
resistance gene to generate the Aurora-A conditional (C) mice. The null allele (-) was
derived by crossing the CN and the C allele harboring mice to ZP3-Cre mice, in which
case the floxed alleles are recombined in the Cre expressing germ cells of females.
PCR Genotyping of mouse tail DNA
PCR genotyping was done utilizing the primer sets listed in the supplementary table.
Genotyping of embryos
Heterozygous mouse breeding pairs were monitored daily for vaginal plugs (0.5 dpc of
embryonic development). At 3.5 or 8.5 dpc, uterus was isolated from plugged females.
For 3.5 dpc embryo genotyping, embryos were flushed with M10 media, then transferred
into M15 media. Each embryo was lysed with lysis buffer (10 mM Tris pH 8.4, 50 mM
KCl, 2 mM MgCl2, 0.45% NP-40, 0.45% Tween 20 and 180 g/ml Proteinase K) at 55oC
for 5 hr and then Proteinase K was heat inactivated at 95°C for 10 min. 5 l of the DNA
lysate were used for PCR.
In vitro growth study of embryos
3.5 dpc embryos flushed with M10 media were transferred onto collagen coated culture
dish (Becton Dickinson) containing ES cell media (DMEM, 15% FBS, 1% L-Glutamine,
1% Penicillin Streptomycin, 1 mM Sodium Pyruvate and 1×MEM non-essential amino
acids), overlaid with oil. Embryos were photographed immediately (3.5 dpc) and after
being incubated for one day (4.5 dpc), two days (5.5 dpc) and three days (6.5dpc) in 5%
CO2 at 37°C by an Olympus 1X70 microscope using 20X objective. Genotyping of the
embryos was done as described above.
Immunofluorescence microscopy
3.5 dpc embryos were flushed with M10 media, and rinsed with PBS. To remove the
zona pellucida, embryos were treated with Acid Tyrode’s, diffused with M15 media, and
then rinsed with M2 media. Embryos were transferred onto poly-L-lysine coated cover
slips (BD Biosciences), incubated with microtubule stabilization buffer (M buffer),
overlaid with modified M buffer (Cutts et al, 1999), and fixed in cold methanol. Embryos
blocked with PBS containing 3% BSA were immunostained with mouse monoclonal
anti-Aurora-A antibody (1:100) and rabbit polyclonal anti--tubulin antibody (Abcam,
1:150). Following incubation with Alexa Fluor 555 goat anti-mouse (Invitrogen, 1:200),
Alexa Fluor 488 goat anti-rabbit (Invitrogen, 1:200) and staining with 4,6-diamidino-2phenylindole (DAPI), the embryos were mounted with SlowFade Gold Antifade Reagent
(Invitrogen) and analyzed by deconvolution microscopy (Nikon 2000U inverted
microscope) using a 4X/1.3 N.A. objectives. Images were acquired and analyzed with a
Photometric CoolSNAP CCD camera (Roper Scientific) and MetaMorph software
(Molecular Devices). Deconvolution processing was performed using AutoDeBlur
software (Molecular Devices).
References:
Cutts SM, Fowler KJ, Kile BT, Hii LL, O'Dowd RA, Hudson DF et al. (1999). Defective
chromosome segregation, microtubule bundling and nuclear bridging in inner
centromere protein gene (Incenp)-disrupted mice. Hum Mol Genet 8: 1145-1155.
McMahon AP, Bradley A. (1990). The Wnt-1 (int-1) proto-oncogene is required for
development of a large region of the mouse brain. Cell 62: 1073-1085.