- Methodology article
- Open Access
Efficient generation of long-distance conditional alleles using recombineering and a dual selection strategy in replicate plates
© Voehringer et al; licensee BioMed Central Ltd. 2009
- Received: 07 January 2009
- Accepted: 28 July 2009
- Published: 28 July 2009
Conditional knockout mice are a useful tool to study the function of gene products in a tissue-specific or inducible manner. Classical approaches to generate targeting vectors for conditional alleles are often limited by the availability of suitable restriction sites. Furthermore, plasmid-based targeting vectors can only cover a few kB of DNA which precludes the generation of targeting vectors where the two loxP sites are placed far apart. These limitations have been overcome in the recent past by using homologous recombination of bacterial artificial chromosomes (BACs) in Escherichia coli to produce large targeting vector containing two different loxP-flanked selection cassettes so that a single targeting event is sufficient to introduce loxP-sites a great distances into the mouse genome. However, the final targeted allele should be free of selection cassettes and screening for correct removal of selection cassettes can be a laborious task. Therefore, we developed a new strategy to rapidly identify ES cells containing the desired allele.
Using BAC recombineering we generated a single targeting vector which contained two different selection cassettes that were flanked by loxP-loxP sites or by FRT-FRT/loxP sites so that they could be deleted sequentially by Cre- and FLPe-recombinases, respectively. Transfected ES cells were first selected in the presence of both antibiotics in vitro before correctly targeted clones were identified by Southern blot. After transfection of a Cre recombinase expression plasmid ES cell clones were selected on replicate plates to identify those clones which maintained the FRT-FRT/loxP flanked cassette and lost the loxP-loxP flanked cassette. Using this strategy facilitated the identification of ES cell clones containing the desired allele before blastocyst injection.
The strategy of ES cell cultures in replicate plates proved to be very efficient in identifying ES cells that had undergone the correct recombination event. This approach facilitates the generation of conditional knock-out mice when large parts of the genome are intended to be flanked by loxP sites.
- Target Vector
- loxP Site
- Conditional Knockout Mouse
- Selection Cassette
- Conditional Allele
Conditional knockout mice are typically generated by insertion of two loxP sites flanking an exon that is critical for gene function. Crossing these mice to mice that express the Cre recombinase in a tissue-specific or inducible manner leads to deletion of the loxP-flanked DNA segment [1, 2]. The classical approach of genetic engineering to generate targeting vectors for conditional alleles can be quite laborious since homology arms and loxP-flanked selection cassettes need to be combined using suitable restriction sites. The development of phage-based recombineering systems which utilize homologous recombination in E. coli have facilitated the generation of suitable targeting vectors (reviewed in ). In addition, the recombineering technology allows the generation of large targeting vectors with the possibility to introduce loxP sites that are separated by several kB of genomic sequence by a single targeting event in ES cells . Here, we modified previously described tools to generate il-4/il-13 double knockout mice. We used a new strategy which facilitates the identification of correctly targeted conditional alleles by culturing ES cells in replicate plates after Cre-mediated deletion of a loxP-flanked resistance cassette.
We generated conditional il-4/il-13 double knockout mice with a single targeting vector that introduced two loxP sites separated by 21 kB of genomic sequence. We used two different selection cassettes with dual pro- and eukaryotic promoters that allowed selection in E. coli and subsequently in ES cells.
First, we retrieved a 27.6 kB segment of genomic sequence from an il-4/il-13 containing BAC clone into the low copy plasmid pBR322 by gap repair recombineering . Fragments of up to 80 kB can be subcloned into pBR322 . At least three reasons argue for subcloning genomic segments from BAC clones. Firstly, the retrieved genomic segments are easier to handle than BACs which grow at only one copy per cell. Secondly, BAC backbones contain a loxP site which should be removed from the targeting vector. Finally, identification of correct targeting events by Southern blot analysis requires that the homology arms flanking the selection cassettes are well defined and not too large.
We describe a new strategy to facilitate the identification of ES cells with correctly recombined conditional alleles. This strategy is based on the use of two different loxP-loxP or FRT-FRT/loxP flanked selection cassettes so that ES cells could be cultured in replicate plates after Cre-mediated removal of the first selection cassette. Southern blot analysis of ES cells that remained resistant to one but lost resistance to the other antibiotic confirmed the correct recombination event. Therefore, this strategy appears very useful for rapid identification of ES cell clones containing the desired allele before blastocyst injection.
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  was transformed with CT7-111I18 BAC and the linearized retrieval vector according to the protocol described previously . 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  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  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 . 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)26Sor tm1(FLP1)Dym ; The Jackson Laboratory, Bar Harbour, ME) to delete the NeoR cassette . 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).
We thank Nigel Killeen and Neal G. Copeland for providing plasmids and cell lines and Judith Johnson for comments on the manuscript. This work was supported by the Howard Hughes Medical Institute, the Sandler Asthma Basic Research Center (to RML) and the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (to DV). The authors have no conflicting financial interests. Correspondence and requests for materials should be addressed to email@example.com.
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