Construction of the Stim1 Knockout Mouse — The cell line, GST050 is publicly available from the Gene
Trap Resource at http://baygenomics.ucsf.edu. The sequence was obtained by 5' RACE PCR from
a mouse ES cell clone with an insertional mutation from a gene trap vector. A portion of DNA
containing the -galactosidase reporter gene with the neomycin resistance gene was inserted
randomly into the genomic DNA (Baygenomics, San Francisco, California). The insertion of the
portion of artificial DNA into the genome disrupts the RNA splicing machinery, resulting in
prevention of protein translation. This insertion can be identified using the activity of the artificial
reporter gene, with the activity of the disrupted gene in mouse tissue also monitored. The Stim1
gene is located on chromosome 7 at site 20137335 with the accession number NM_009287.4. The
ES cell line, GST050, transfected with the gene-trap vector pGT1TMpfs (BayGenomics, University
of San Francisco, San Francisco, California) contains the first 3 exons of the Stim1 gene with the
disruption vector containing -galactosidase-neomycin inserted at 124 aa of the mRNA,
corresponding to 120847 bp of the Stim1 gene in Intron 3-4 of the genomic DNA as identified by
using partial Inverse PCR (see below). The BayGenomics ES cell line was generated from 129P2
(formerly 129/Ola) ES cell lines {Nichols, 1990 #8588}). Parental cell lines (CGR8 and E14Tg2A)
were established from delayed blastocysts on gelatinized tissue culture dishes in ES cell medium
containing LIF {Nichols, 1990 #8588}. Sub-lines were isolated by plating cells at a single-cell
density, picking, and expanding single colonies, and testing several clones for germ-line competence.
The single clone ES cell line was passaged for six days in ES cell medium (GMEM, 2 mM glutamine,
1 mM sodium pyruvate, 1 X non-essential amino acids, 10% FCS, 1:1000 dilutionmercaptoethanol, and 1000 U LIF (leukemia inhibitory factor - necessary for the maintenance of
undifferentiated embryonic stem cells) in the absence of neomycin. On the day of injection, the
medium was changed several hours before harvesting the cells. A confluent 25 cm 2 flask was
trypsinized for 4 minutes and diluted into 9 mL of cold ES cell medium without LIF, pelleted, and
resuspended in 0.8 mL of ES cell medium (without LIF) in a sterile 1.5 mL screw-top
microcentrifuge tube. Before they were added to the injection chamber, cells were kept on ice (for
up to several hours) to prevent clumping. Blastocysts were flushed from pregnant C57BL/6 females
and collected into a CO2-independent medium containing 10% FCS. Blastocysts were expanded for
one to two hours in ES cell medium in a 37°C/5% CO2 incubator, transferred to a hanging drop
chamber, and cooled to 4°C. ES cells were added to the hanging drops, and the blastocysts are
injected with enough cells (20 or more) to fill the blastocoele. Injected blastocysts were then
transferred to pseudo pregnant recipient females (10-15 blastocysts/uterine horn). Typically,
injection of 10 blastocysts will yield an average of twice male chimeras with germline mosaicism.
These ES cells were microinjected into 3.5-d-old C57BL/6J blastocysts to generate chimeric mice.
Chimeric males were analyzed for germline transmission by mating with C57BL/6J females, and the
progeny were analyzed by PCR analysis, -galactosidase staining, and Southern blot analysis (See
below). Deficient mice were generated in the HSLAS facility at the University of Alberta with the
assistance of Dr. P. Dickie.
To identify where the Stim1 gene was disrupted, partial Inverse PCR was utilized (Laurent
Vergnes and Karen Reue). This involved using specific primers against the 3’ end of the gene-trap
disruption vector, digesting the DNA, ligating the DNA into small circular DNA fragments and
performing nested PCR. Genomic DNA was isolated from -galactosidase staining identified
heterozygote tail clippings and the integrity of the vector was identified by using standard PCR with
one specific forward primer and three reverse primers to determine whether the 3’ end of the vector
had been degraded, which is an unfortunate common occurrence in gene trapping. The sequence of
the gene-trap vector was downloaded from the BayGenomics website.
Forward primer:
Reverse primer 1:
pGTCF
pGTCR1
5’-GGTCTGACGCTCAGTGGAACG-3’
5’-GGGATCATGTAACTCGCCTTG-3’
Reverse primer 2:
Reverse primer 3:
pGTCR2
pGTCR3
5’-GAAGATCAGTTGGGTGCACG-3’
5’-CATCCCCCTTTCGCCAGCT-3’
Standard PCR identified partial degradation occurring at the 3’ end of the plasmid downstream of
the third primer pGTCR3. This dictated the use of Inverse PCR using specific primer set B, INVF3,
INVR3, INVF4, and INVR4.
INVF3, GTACTGAGAGTGCACCATATGC
INVR3, AGTTAAGCCAGCCCCGACACC
INVF4, GCACAGATGCGTAAGGAGAA
INVR4, ACCCGCCAACACCCGCTG
Genomic DNA was digested with BfaI, an enzyme that has multiple sites of cleavage in
genomic DNA as well as the vector, digesting the DNA into small fragments, one of which
hopefully contains a portion of the disruption vector followed by a portion of the calnexin gene.
The DNA is purified using a PCR purification kit (Qiagen) and the small fragments are ligated using
the Rapid DNA Ligation Kit (Boehringer) to generate small circular DNA fragments. Inverse PCR
was done using 20 ng of ligation reaction and primer set, INVF1 and INVR1. Product was purified
using QIAquick Gel Extraction Kit (Qiagen). If no band is observed, a small portion of the PCR
product was used to perform a nested PCR using the primer set, INVF3 and INVR3. After the
second PCR, the product was purified and send for sequencing. The sequence obtained contained
gene-trap vector followed by sequence specific to the Stim1 gene, identifying the insertion site of the
gene-trap vector as bp 2451 of Intron 3-4. Using this information, primers were designed that
utilized the inserted gene-trap vector as a forward primer, INVF3 and a reverse primer, Stim1R5,
located in Intron 3-4 of the Stim1 sequence to detect the disrupted strand, giving a PCR product of
300 bp. To detect wild type strand, a forward primer Stim1F4 and Stim1R6 were designed that
correspond to either side of the insertion site in Intron 3-4, giving a PCR product of 1100 bp.
I “walked” down intron 3-4 with primers to identify where the stim1 gene was disrupted and send PCR
product for sequencing:
pGT1.8TM disruption vector
linker region
Stim1 intron 3-4
Ttgtaaaacgacggccagtgccaagctt
ccggtacatgatcgaggggactgacaagacgg
cctcccgcagttaaatgttaaa…
To Identify disruption I used two sets of primers with genomic DNA:
Genotype genomic knockout strand with:
INVF3, GTACTGAGAGTGCACCATATGC
Stim1R5 5’-cagtagacagcctgcattgc-3’
Size: 300 bp
Genotype genomic wild type strand with:
Stim1F4 5’-cagtgcacttccttgtcagctg-3’
Stim1R6 5’-ccgtctatgactccatggcag-3’
Size: 1100 bp
Protein Product:
14 kDa fragment + 178 kDa b-gal Engrailed fragment:
124 aa:
M D V C A R L A L W L L W G L L L H Q G Q S L S H S H S E K N T G A S S G A T S E E
S T E A E F C R I D K P L C H S E D E K L S F E A V R N I H K L M D D D A N G D V D V
E E S D E F L R E D L N Y H D P T V K H S T F H G E D K L I S V E D L W K A W K S S E
1-22: signal sequence
23-213: luminal domain
214-234: transmembrane domain
132-200: SAM domain
63-98: EF hand
76-87: Calcium binding domain
248-388: Coiled Coil domain
Sequence of Stim1 that will still be present:
ATGGATGTGTGCGCCCGTCTTGCCCTGTGGCTTCTTTGGGGGCTCCTTCTGCATCAGGGCCAGAGTCTCAGCCATA
GTCACAGTGAAAAGAATACAGGAGCTAGCTCCGGGGCGACTTCTGAAGAGTCTACCGAAGCAGAGTTTTGCCGAA
TTGACAAGCCCCTGTGCCACAGTGAGGATGAGAAGCTCAGCTTTGAGGCCGTCCGAAACATCCATAAGCTGATGG
ATGACGATGCCAATGGTGATGTGGATGTGGAAGAAAGTGATGAGTTCCTAAGGGAAGACCTCAATTACCATGACC
CAACAGTGAAACATAGCACCTTCCATGGTGAGGATAAGCTTATCAGCGTGGAGGACCTGTGGAAGGCGTGGAAG
TCATCAGAA