Supplementary Methods
Embryo isolation, preparation and screening of single cell cDNAs
LS and EB stage 129/SvEv embryos were isolated at E7.0 and E7.5, respectively, in
DMEM with 10% fetal calf serum buffered with 25mM HEPES, pH7.4, at room
temperature. Preparation and quantification of single cell cDNAs by Southern blotting
was performed essentially as described previously1,2 (Fig. 1). For single cell
comparisons between mutant and control embryos (Fig. 6), MB to EHF E7.5 embryos
were used, where mutant embryos were generated from Blimp-1 (-/-) ES cells (see
below), while loxP/loxP ES cell derived or normal wild type embryos were used as
controls to isolate single cells. Single cell cDNAs were generated using 30 cycles of
PCR-amplification, followed by an additional 30 PCR cycles to determine expression
of specific genes.
In situ hybridization, antibody staining, and alkaline phosphatase staining
In situ hybridization was performed as described previously3. For some experiments,
posterior-proximal extraembryonic mesoderm was isolated and disaggregated to
determine the number of stained cells. Antibody stainings on dispersed E7.5 germ cell
regions and on whole embryos were carried out essentially as described previously 4,5.
Antibodies used were: rabbit anti-PGC7/Stella (1:2000, gift from T. Nakano),
monoclonal rat anti-GFP (1:500, Nacalai Tesque Inc., Kyoto), rabbit anti-GFP
Alexa488 conjugated (1:500, Molecular Probes, Eugene, OR), donkey anti-rabbit IgG
Alexa594 conjugated (1:500, Molecular Probes), goat anti-rabbit IgG Alexa488
conjugated (1:500, Molecular Probes) and goat anti-rat IgG Alexa488 conjugated
(1:500, Molecular Probes). PGC counts following TNAP-staining was performed as
described previously6,7 .
BAC modification and transgenic mice production
To create Blimp-1mEGFP reporter mice, EGFP coding sequence was subcloned into
the BglII and SalI site of the pDisplay vector (Invitrogen). The resulting membranetargeted cassette of EGFP with a polyadenylation signal was amplified and
recombined into just after the methyonine of exon 3 of Blimp-1 gene in a 230 kb
bacterial artificial chromosome of C57BL/6 backgound bearing 160kb upstream and
70kb downstream region from the Blimp-1 transcription start site purchased from
BACPAC Resources Center (http://bacpac.chori.org/), according to the protocol
supplied by GeneBridges (http://www.genebridges.com). The modified BAC was
injected into male pronuclei of oocytes from C57BL/6/DBA mixed background to
generate transgenic mice. Transgenic embryos were imaged on an inverted ZeissLSM 510 confocal microscope. Image-processing and cell counts were performed
using Zeiss LSM Image Browser. To create Blimp-1-Cre line, BAC clone #452 P8
(Research Genetics) containing Blimp-1 locus was modified using a PCR-based
method8. The coding region in the first exon of Blimp-1 was substituted by the first
100 bp of the Cre sequence preceded by an NLS. The second exon of Blimp-1 was
replaced by the remaining Cre sequence followed by a transcriptional STOP cassette.
The modified BAC was linearized and microinjected into pronuclei of zygotes
derived from B6CBAF1 mice.
Generation of Blimp-1 knockout cells, mice and tetraploid chimeras
The Blimp-1 targeting vector (pDTA-TK-Blimp-1) was engineered to flank exon 5
with loxP sites and then transfected into E14.1 ES cells (129/ola). Out of 200 ES
clones, four clones had one homologously recombined Blimp-1 allele and one clone
had both alleles targeted (Clone 2G3). After Cre-mediated deletion of the loxPflanked neor gene, heterozygous mice for the Blimp-1 loxP allele were generated.
These mice were bread to deleter-Cre9 to generate the Blimp-1 null allele. The
resulting Blimp-1 (+/-) mice where kept on a mixed C57BL/6/CBA/129 background
and intercrossed to analyze embryos for PGC-development. To make Blimp-1 (-/-) ES
cells, we electroporated double targeted ES cells (clone 2G3) with PGK-Cre plasmid
(gift from Sam Aparicio). We identified several -/- clones, of which two were injected
into Rosa 26-lacZ10 tetraploid host blastocysts to generate ES cell derived embryos as
described previously11. We also got one loxP/loxP clone (2-2A), which was used as a
control.
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