Supplemental Data

Files in this Data Supplement:

• [Supplemental Table 1] - "List and sequences of the designed specific primers for PCR studies."
• [Supplemental Figures] - "Supplemental Figure 1. Targeting construct and targeted disruption of the Ldhc gene. Earlier attempts to generate a targeted disruption in the Ldhc gene were unsuccessful because it was not known at the time that 43% of the intronic sequences of the gene consist of repetitive elements [1]. Care was taken in the present study to target recombination sites to sequence regions free of repetitive elements for a construct that would allow deletion of exon 3, which encodes the coenzyme-binding domain and part of the substrate-binding domain. The targeting construct was derived from a 129SvEv genomic clone (PAC257) and the nucleotide numbers indicated are based on the 20005 bp DNA Ldhc genomic sequence AF190799 (GI 10802595). The backbone plasmid was a modified (pBluescript KSII MCS sites) pBR322 vector designated pBR25F and the targeting construct was made by reassembling regions of the Ldhc genomic DNA with vector MCS/floxed components. A 2.8 Kb PvuII digested fragment (bp 2945 to 5764) was used as the 5’ homologous arm (A). A 2.8 Kb PstI and BstXI digested fragment (bp 5802 to 8637) which included exon 3 was used as targeting fragment (B). The 2.5 Kb BamHI digested fragment (bp 8710 to 11258) was used as the 3’ homologous arm (C). A small region (31 bp) was excised between fragments A and B and a loxP site was inserted at that location. The Neo gene flanked with loxP sites was inserted between fragments B and C, replacing 71 bp of intronic sequence, to allow positive selection. A tk gene was placed after fragment C to allow negative selection. The targeting construct was verified by PCR, sequencing, and restriction digestion. The vector was grown in host bacteria Stb14 (Invitrogen).

  The NotI linearized construct was electroporated into TC1 embryonic stem (ES) cells (gift of Dr. Philip Leder, Harvard Medical School) using a Bio-Rad Gene Pulser. Electroporation, selection, colony isolation, and homologous recombination determination were performed as described previously [2]. Drug-resistant ES cell colonies were verified for correct targeting by PCR and Southern analysis and cryopreserved for later use. The 3' PCR screening primers used to detect homologous recombination (3305 bp band) were forward 5'-GCC TAC CCG CTT CCA TTG CTC-3' and reverse 5'-GTG GGC TCT CCT GCT CTC ATC ACT-3', corresponding to sequences in the Neo gene and 3’ flanking region, respectively. The 5’ PCR screening primers used to detect homologous recombination (6739 bp band) were forward 5'-TGA GGC TGG GCT GGA CTA TGT GA-3'and reverse 5'-GGG GGT GGG GTG GGA TTA GAT AAA-3', corresponding to sequences in the 5’ flanking and Neo gene, respectively.

  For Southern blot analysis (not shown), 15 to 20 &[micro]g of ES cell DNA was extracted, purified, and cut with a restriction enzyme that could distinguish wild type and targeted alleles based on size (ApaI or Tth111I), resolved on 0.8% agarose gels, transferred to nitrocellulose membrane and hybridized with a randomly labeled radioactive internal probe, or cut with BamHI or AflII, and hybridized with a flanking probe.

  Approximately 10-15 ES cells were injected into C57BL/6N blastocysts and the blastocysts were surgically implanted in surrogate females. Male chimeras with a high percentage 129SvEv contribution as judged by coat color were mated with C57BL/6N females to determine germline transmission. Animals carrying the floxed allele (Type 1; Ldhc^f) were backcrossed onto the C57BL/6N genetic background. Males and females with one (Ldhc^f/+) or two (Ldhc^f/f) floxed alleles were produced for fertility evaluation. No difference with wild type was observed (data not shown).

  Transgenic MMTV-Cre/line A [B6129-Tg(MMTV-Cre)1Mam] mice obtained from Jackson Laboratory have been backcrossed repeatedly with C57BL/6N mice in our laboratory since May 2001. This strain (stock number 003551) expresses Cre recombinase in the female germline under the control of the MMTV LTR promoter. Type I (Ldhc^f/+) males were mated with hemizygous MMTV-Cre females and female offspring carrying both the floxed (Ldhc^f/+) gene and the Cre transgene were mated with C57BL/6N males to produce offspring with a deletion of exon 3 in one copy of the Ldhc gene (Type 4; Ldhc^+/-). The Ldhc^+/- mice were backcrossed with C57BL/6N mice to establish colonies carrying the mutation. Pairs of Ldhc^+/- mice produced in these colonies were mated to produce wild type Ldhc^+/- and Ldhc^-/- offspring.

  Supplemental Figure 2. Immunohistochemistry of ovary from wild type (WT), Ldhc^+/- (HET) and Ldhc^-/- (KO) female mice. Arrows indicate positive areas and the asterisk indicates the absence of LDHC.

  "

• [Supplemental Movie 2] - "Ldhc^-/- (KO) sperm after 60 min incubation in capacitating medium."
• [Supplemental Movie 1] - "Ldhc^+/- (HET) sperm after 60 min incubation in capacitating medium."
• [Supplemental Movie 4] - "Ldhc^-/- (KO) sperm after 4 h incubation in capacitating medium."
• [Supplemental Movie 3] - "Ldhc^+/- (HET) sperm after 4 h incubation in capacitating medium."