Generation of the targeting vector
The retrieval vector was constructed by cloning two short homologous arms, generated by PCR amplification of CT7-111I18 BAC DNA (from CitbCJ7 mouse BAC library; 129Sv origin) with the following primer sets: 5'RV-1: gtcgacgcggccgcactagtttgtgtatattg and 5'RV-2: gaattcctagaactctgtagatcag for the 5' homolgous arm and 3'RV-1: gaattcacaccaacactgacatc and 3'RV-2: ggatccggaccgcccttaatccacgcg for the 3'homologous arm. These arms were first cloned into pCR2.1-TOPO and sequenced, then cut with Sal I/EcoR I (5'RV) or BamH I/EcoR I (3'RV) and subcloned into BamH I/Sal I digested pBR322 which had been cut with EcoR I and Hind III, blunted and religated to destroy the EcoR I site. A full length diphtheria toxin alpha counter-selection cassette was then cloned by RsrII digest from pKO SelectDT (Lexicon Genetics, TX) into the Rsr II site present in primer 3'RV-2 to generate the final retrieval vector (Figure 1A). The E. coli strain DY380 [5] was transformed with CT7-111I18 BAC and the linearized retrieval vector according to the protocol described previously [4]. In brief, BAC-transformed DY380 cells were heat-induced by incubation at 42°C for 15 minutes, chilled on ice for 5 minutes, washed in ice-cold water and electroporated with 10 ng EcoR I linearized retrieval vector using a Bio-Rad electroporator set to 1.75 kV, 25 μF, 200 Ω. Recombinant clones were selected at 30°C on LB-Amp plates.
MTVs were constructed by PCR amplification of CT7-111I18 DNA using the following primer pairs: for the 5'MTV: primers A: 5'-tggcggccgctattggctgttggcttc-3' and B2: 5'-gagaattctgtagttaagggtcac-3'; and primers C: 5'-gaggatccttcctcatgctgtggtg-3' and D: 5'-gagtcgacgaaggccgttgaactg-3'. For the 3'MTV: primers E: 5'-aagcggccgctaacacagtagaactac-3' and F: 5'-ggaattcatgtcttgatctgaaag-3'; and primers G: 5'-gaagatctgacacagtgcctctg-3' and H: 5'-ggagtcgactggctggcctggagctc-3'. To complete the 5'MTV which was designed to target the second intron of il-13, PCR products AB2 and CD were cut with Not I/EcoR I and BamH I/Sal I, respectively, and cloned together with a EcoR I/BamH I digested loxP flanked blasticidin-resistance cassette (BsdR) into Not I/Sal I digested pBluescript. The BsdR cassette was generated by exchange of the original neomycin/kanamycin resistance cassette (NeoR) in plasmid PL452 [4] for a BsdR gene isolated by Nco I/Bcl I digest from pCoBlast (Invitrogen). The 3'MTV was assembled by cloning PCR products EF and GH, digested with Not I/EcoR I and Bgl II/Sal I, respectively, together with EcoR I/BamH I digested FRT-FRT/loxP-flanked NeoR cassette from plasmid PL451 [4] into Not I/Sal I digested pBluescript.
The 3'MTV was electroporated into heat-induced DY380 cells containing the retrieved BAC subsequence and colonies were selected on LB-kanamycin plates. After the targeting event was confirmed by PCR, cells were again heat-induced and electroporated with 5'MTV and selected on LB-kanamycin/blasticidin plates to generate the final targeting vector used for homologous recombination in ES cells.
Targeting of ES cells and Cre-mediated deletion of the BsdRcassette
107 E14 ES cells (129/Ola background) were electroporated with 30 μg Not I-linearized targeting vector and clones were selected in G418 (0.25 mg/ml) and blasticidin (10 μg/ml) containing medium. 240 clones were picked and subjected to Southern blot analysis. Genomic DNA was digested with EcoR I, run on a 1% agarose gel, blotted, hybridized first with the 3'probe, then stripped and hybridized with the 5'probe. The probes were generated by PCR using CT7-111I18 DNA as template and primers 5'probe1: 5'-gtcacgagccagaccattcg-3' and 5'probe2: 5'-cactcatgagcccacagc-3' and primers 3'probe3: 5'-gagagaggaactctgggatag-3' and 3'probe4: 5'-gctgcagcaggactctactg-3'. The 3'probe shows a band at 7.4 kB for the wild-type allele and 5.5 kB for the targeted allele. The 5'probe shows a band at 17.5 kB for the wild-type and 10.4 kB for the targeted allele.
To delete the BsdR cassette 107 cells of an ES cell clone was electroporated with 10 μg supercoiled pMC-CreN plasmid which expresses the Cre-recombinase with a SV40-derived nuclear localization signal sequence under control of HSV-tk promoter/enhancer elements [17]. 1000 cells were seeded on a 10 cm dish and grown in medium containing only G418. 120 subclones were picked and grown in replicate plates, one only with G418 selection, the other one with G418 and blasticidin to identify clones that lost the ability to grow in the presence of blasticidin. 8 of 120 subclones had lost the ability to grow under blasticidin selection and were subjected to Southern blot analysis. Genomic DNA was digested with BamH I, run on a 1% agarose gel, blotted and hybridized with the 5'probe. The wild-type allele generated a band at 6.5 kB and the correctly recombined allele a band at 4.8 kB.
Generation of mice and deletion of the NeoRcassette
ES cells were injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 mice and offspring were analyzed by PCR for germ line transmission. Mice that contained the targeted allele were bred with FLPe deleter mice (Gt(ROSA)26Sortm1(FLP1)Dym; The Jackson Laboratory, Bar Harbour, ME) to delete the NeoR cassette [10]. Successful deletion and correct targeting at both sites was demonstrated by Southern blot analysis after digestion of tail DNA with EcoR I and hybridization with the 3'probe which resulted in bands at 7.4 kB (wild-type allele) and 3.6 kB (Neo-deleted allele) or by BamH I digest and hybridization with the 5'probe resulting in bands at 6.5 kB (wild-type) and 4.8 kB (targeted allele) (Figure 3B).
In addition, both targeting events were confirmed by PCR using primers A2: 5'-cagcatggtatggagtgtg-3' and D2: 5'-cattgcaattggagatgttg-3' or primers I: 5'-cttgaatacttggtccaccg-3' and 3'4PCR-2: 5'-gaaacaggttctcattatgtag-3' (Figure 3C).
Th2 cell polarization, mast cell culture and RT-PCR analysis
CD4 T cells from spleen and lymph nodes of CD4-Cre/4–13F/- or 4–13F/- mice were purified by negative selection using the MACS CD4 T cell isolation kit (Miltenyi Biotec, Germany) according to manufacturer's instructions and cultured for 5 days in the presence of 20 ng/ml IL-2, 20 ng/ml IL-4 and 20 μg/ml anti-IFN-γ to induce Th2 polarization. Bone marrow cells were cultured for 11 days in the presence of 3 ng/ml IL-3 and 3 ng/ml SCF to generate mast cells. RT-PCR was performed with cDNA from Th2 and mast cell cultures using the primers: IL-4fwd: 5'-agctagttgtcatcctgctc-3' and IL-4rev: 5'-tggtggctcagtactacgag-3', IL-13fwd: 5'-gcagtcctggctcttgcttg-3' and IL-13rev: 5'-tgctttgtgtagctgagcag-3', hprt-fwd: 5'-gttggatacaggccagactttgttg-3' and hprt-rev: 5'-gagggtaggctggcctataggct-3'. PCR reactions were performed with 58°C annealing temperature and 60 sec extension time at 72°C using the SYBR® green Taq ReadyMix™ (Sigma) and Lightcycler PCR machine (Roche, Switzerland).
Experiments with animals
All mice were housed in the specific pathogen-free animal facility at UCSF according to institutional guidelines. The experiments have been approved by the Institutional Animal Care and Use Committee (IACUC).