Blood, Vol. 114, Issue 12, 2401-2410, September 17, 2009

Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis
Blood Stumpo et al. 114: 2401

Supplemental materials for: Stumpo et al

Construction of targeting vector and generation of knockout (KO) mice
A 129SV mouse genomic library in lambda FIX (Stratagene, La Jolla, CA) was screened by standard techniques 1 with a 275 bp PstI fragment from an EST clone (GenBank accession number AA021952.1).2 A 7.5 kb XbaI fragment containing Zfp36l2 was isolated and subcloned into pBluescript (Stratagene, La Jolla, CA) to create the plasmid pSK/Zfp36l2-7.5. The plasmid pPGKneobpAlox2PGKDTA, pNLD (generously provided by Philip Soriano, Fred Hutchinson Cancer Research Center), containing the positive selection marker Neomycin (Neo) and the negative selection marker diphtheria toxin (DTA), was used as a backbone for the Zfp36l2 targeting vector. A 5.2 kb XbaI/SalI fragment (long arm) was excised from pSK/Zfp36l2-7.5, blunted and ligated with blunted, NotI digested pNLD. This plasmid, designated pNLD/Zfp36l2-5.2, contained the 5′ portion of Zfp36l2. To extend the 3′UTR of the original Zfp36l2 7.5 kb genomic clone using 3′ RACE, RNA isolated from wild-type (WT) mouse bone marrow-derived macrophages was used as a template for reverse transcription using an oligo-dT primer. Reverse transcription was performed with Superscript II Rnase H− (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The 3′UTR was amplified using the following PCR primers: forward, 5′-ACGTGGTACCGCCATTATTAATCGGCAAAATGTGG-3′ (corresponding to nucleotides 2639 to 2663 of GenBank accession number NM_001001806.2 with the inclusion of an Asp718 (underlined) site); reverse, 5′-GATCGGATCCCAATTAACCCTCACTAAAGGG(T)18VN-3′ (anchored oligo(dT) primer). The amplified product was about 950 bp in size and contained the polyadenylation signal. A 724 bp Csp45I fragment was isolated from this amplified product and ligated with Csp45I/ClaI digested pSK/Zfp36l2-7.5, creating a new Zfp36l2 genomic clone containing the entire 3′UTR of the gene, pSK/Zfp36l2-8.2. A 1.4 kb NotI/Csp45I fragment (short arm, corresponding to nucleotides 1408 to 2803 of NM_001001806.2) was excised from pSK/Zfp36l2-8.2, blunted and ligated with blunted, SalI digested pNLD/Zfp36l2-5.2, generating the final Zfp36l2 targeting vector, pNLD/Zfp36l2. This targeting vector resulted in the deletion of the majority of the protein coding region, bases 257 through 1242 of NM_001001806.2.

A control plasmid was created for PCR screening of electroporated ES cells that would allow for the identification of homologous recombination events. A 2.17 bp NotI/Asp718 fragment (corresponding to nucleotides 1408 to 3537 of NM_001001806.2) was excised from pSK/Zfp36l2-8.2, blunted and subcloned into SalI digested, blunted pNLD. This construct contained 0.77 kb of sequence downstream of the targeting vector and was designated pNLD/Zfp36l2-2.17. All PCR products, newly generated plasmids and the final targeting vector were sequenced with the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit or the Big-Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).

The targeting vector, pNLD/Zfp36l2, was linearized with Asp718 and electroporated into AB2.2 embryonic stem (ES) cells (Lexicon Genetics, Woodlands, TX) using standard techniques.3 Genomic DNA from electroporated ES cells was first screened for homologous recombination using PCR. PCR conditions were as previously described 4 with the exception that an annealing temperature of 62°C was used. PCR primers used for this screening were: PGKNeo forward (F1; corresponding to nucleotides 2035 to 2048 of the neomycin gene in pPGKneobpAlox2PGKDTA), 5′-ATTCGCAGCGCATCGCCTTCTATC-3′; reverse (R1; corresponding to nucleotides 2968 to 2949 of NM_001001806.2), 5′-TGGATGTAGAGAAGCAAGCC-3′. These primers would result in a 2 kb product only if homologous recombination had occurred. Potential positive homologously recombined ES cell clones were subsequently confirmed by Southern analysis. Genomic DNA was isolated from ES cell clones and digested with EcoRV and SstI (5′ end) or with EcoRI (3′ end), separated on a 0.7% agarose gel, transferred to Hybond+ (GE Healthcare, Piscataway, NJ) and hybridized to random-primed α-32P–labeled 5′ and 3′ probes. The probe used to detect homologous recombination at the 5′ end of the gene was a 286 bp XbaI fragment excised from the original Zfp36l2 genomic clone, pSK/Zfp36l2-7.5, and the probe used to detect homologous recombination at the 3′ end of the gene was a 724 bp Csp45I fragment excised from pSK/Zfp36l2-8.2. Four correctly targeted ES cell clones were identified out of 1728 total G418-resistant ES cell clones. Two of these clones were injected into C57BL/6J-TyrC-2J blastocysts and used to generate chimeric mice according to standard procedures.3 Male chimeras were mated to C57Bl/6 females and heterozygous (Het, +∕−) offspring were intercrossed to generate homozygous knockout (KO, −∕−) mice (B6;129-Zfp36l2tm2Pjb). Genomic DNA was isolated from mouse tail pieces as previously described5, and 50 ng was used as a template for PCR. The primers used were: PGKNeo forward (F1; corresponding to nucleotides 2035 to 2048 of the neomycin gene in pPGKneobpAlox2PGKDTA), 5′-ATTCGCAGCGCATCGCCTTCTATC-3′; Zfp36l2 forward (F2; corresponding to nucleotides 1380 to 1401 of NM_001001806.2), 5′-CCATCCAGACCCACAACTTCGC-3′; and Zfp36l2 reverse (R2; corresponding to nucleotides 1861 to 1838 of NM_001001806.2), 5′-AACGAGAGAACCGAAAGGGTAGGG-3′. These primers amplified a 482 nucleotide fragment from the endogenous Zfp36l2 gene and an 898 nucleotide fragment from the Neo-interrupted Zfp36l2 gene. The initial offspring from heterozygous intercrosses were genotyped by Southern analysis as described above for the targeted ES cells and, once confirmed to be correctly targeted, routine genotyping was done by PCR.

Isolation of MEFs and mRNA decay studies
Mouse embryo fibroblasts (MEFs) were isolated from fetal mice at embryonic day (E) 14.5 of gestation, where E0.5 was the date of detection of the vaginal plug. MEFs were prepared from individual fetuses as previously described.6 Genotypes were determined from DNA isolated from the tails of the fetuses. Cells were maintained at 37°C (5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% (vol/vol) fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Cells were passaged as described previously,7 and were used at passage 3 for these experiments.

mRNA decay studies were done with serum-deprived MEFs. Cells that were at approximately 70 to 80% confluence were washed once in serum-free DMEM and then incubated for 16 hours in DMEM containing 0.5% (vol/vol) FBS. Cells were stimulated for 60 or 90 min with 10% FBS (Hyclone, Logan, UT) followed by the addition of actinomycin D (Sigma Chemical Co., St. Louis, MO) at a final concentration of 5 µg/µl. Cells were harvested at the indicated times after the addition of actinomycin D, and RNA was isolated as described above using either the Qiagen RNeasy or the GE Healthcare Illustra RNAspin kit.

Affymetrix microarray hybridization
Gene expression analysis was conducted using Affymetrix Mouse Genome 2.0 GeneChip® Arrays (Mouse 430 v2, Affymetrix, Santa Clara, CA). One µg of total RNA was amplified as directed in the Affymetrix One-Cycle cDNA Synthesis protocol. Fifteen µg of amplified biotin-cRNAs were fragmented and hybridized to each array for 16 hours at 45°C in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed using the EukGE-WS2v5 protocol of the Affymetrix Fluidics Station FS450 for antibody amplification. Arrays were scanned in an Affymetrix Scanner 3000 and data were obtained using the GeneChip® Operating Software (GCOS; Version 1.4.0.036). Data preprocessing, normalization, and error modeling were performed with the Rosetta Resolver system (Version 7.0).

TaqMan® real-time PCR and statistical analysis
The levels of selected transcripts identified by the microarray analysis were measured with TaqMan® real-time PCR. Isolated RNA was reverse transcribed into cDNA using ABI’s High Capacity cDNA Archive Kit (Applied Biosystems). Each Taqman® reaction was performed as follows: 3 µl of cDNA, 12.5 µl of Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 1.25 µl of pre-designed primer probe set (Applied Biosystems, Foster City, CA), and 8.25 µl of H2O. All reactions were performed in triplicate in 96-well plates. Each TaqMan® PCR reaction was performed under the following conditions: 2 min at 50°C followed by 95°C for 10 min, then 40 cycles at 95°C for 15 sec, then 60°C for 60 sec, using the ABI/Prism 7900HT Sequence Detector System. The Cxcl1 primer/probe set used was a pre-designed primer/probe set commercially available from ABI Assay-on-Demand™ gene expression products (Mm00433859_m1). Results were normalized to the endogenous control GAPDH, and the fold change was calculated using the 2−ΔΔCt method of relative quantification.8 Student’s t test (two samples assuming equal variances, two-tail) was used to determine significant differences between KO and WT groups. A p value of

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