The
Hdac2 mutant mice were generated by homologous recombination in embryonic stem (ES) cells where exons 2 to 5 of the
Hdac2 gene encoding a portion of the histone deacetylase domain were deleted (
Fig. 1A). The
Hdac2 targeting vector was derived using long-range PCR to generate the 5′ and 3′ arms of homology using 129S5 ES cell DNA as a template. The 2536 bp 5′ arm was generated using primers
Hdac2-3 [5′-ATGGATCCTATGACAGAAGTTTGTCGAAAGCTG-3′] and
Hdac2-SfiA [5′-ATGGCCGCTATGGCCTGAGGCTTCATGGGATGACCCTGG-3′] and cloned using the TOPO (Invitrogen, Grand Island, NY) cloning kit. The 2718 bp 3′ arm was generated using primers
Hdac2-SfiB [5′-ATGGCCAGCGAGGCCCCTGCAGAAGTGTGAGAAATAGACC-3′] and
Hdac2-6 [5′-ATGGTACCAGCAGCATTCAGTGGGTCCACATG-3′] and cloned using the TOPO cloning kit. The 5′ arm was excised from the holding plasmid using BamH-I and Sfi I. The 3′ arm was excised from the holding plasmid using Sfi I and Kpn I. The arms were ligated to a Sfi I prepared selection cassette containing a β-galactosidase-neomycin marker and inserted into a BamH-I/Kpn I cut pKO Scrambler vector (Stratagene, Santa Clara, CA) to complete the
Hdac2 targeting vector, which results in the deletion of coding exons 2 to 5. The Not I linearized targeting vector was electroporated into 129S5 ES cells (Lex2). G418/FIAU-resistant ES cell clones were isolated, and correctly targeted clones were identified and confirmed by Southern blot analysis using a 297 bp 5′ external probe (14/15), generated by PCR using primers
Hdac2-14 [5′-CACCCTAACACCATTACATTG-3′] and
Hdac2-15 [5′-ACAACACATTCCAAGGACTTG-3′] and a 262 bp 3′ external probe (12/13), amplified by PCR using primers
Hdac2-12 [5′-GCATAGGTTGTCTTGGGTAG-3′] and
Hdac2-13 [5′-TCCTCAGAGCTTCTACATCC-3′]. Southern blot analysis using probe 14/15 detected a 19.0 Kb wild-type band and 11.7 Kb mutant band in BamH-I-digested genomic DNA, while probe 12/13 detected a 7.5 Kb wild-type band and 5.5 Kb mutants in Nde I-digested genomic DNA. Correctly targeted clones (
Fig. 1B) were used to generate heterozygous mutant mice. Two targeted ES cell clones were identified and microinjected into C57BL/6 (albino) blastocysts to generate chimeric animals, which were bred to C57BL/6 (albino) females, and the resulting heterozygous offspring were interbred to produce homozygous
Hdac2-deficient mice. Determination of the genotype of mice at the
Hdac2 locus was performed by extracting and screening DNA from tail biopsy samples using quantitative PCR and KAPA2G Fast HotStart Genotyping Mix (KaPa Biosystems, Inc., Woburn, MA) for the
Neo cassette (
Fig. 1C). This strategy enabled discrimination of zero, one, or two gene disruptions representing
Hdac2 +/+,
Hdac2 +/−, and
Hdac2 −/− mice, respectively. Age-matched wild-type littermates were used as control. We found that most of the
Hdac2-null mice could not survive long enough for most of our experiments. This is consistent with another study reporting that
Hdac2-null mice died early after birth due to severe cardiac malformations.
21 Therefore,
Hdac2 heterozygous knockout mice were used in the majority of the current studies. Animals were reared under cyclic light (12 hours light/12 hours dark) with ambient light intensity. Mice aged 10 to 12 weeks were used for experiments.