On a vitamin B12-deficient diet Cd320 KO mice die of a severe anemia starting around 10 months of age. Female Cd320 KO mice fail to reproduce with fetal death prior to fetal implantation in the uterus.

The Cd320 gene was targeted in mouse embryonic stem (ES) cells using the Cre-loxP recombination system. A targeting vector was introduced into HGTC-8 ES cells (derived from the C57BL/6J strain) via electroporation, employing a positive/negative double-selection strategy that yielded a 1:6 enrichment of doubly selected clones. From 240 cloned ES cell colonies, correctly targeted clones were identified through Southern blot analysis. Two validated clones were expanded and microinjected into BALB/C blastocysts, resulting in founder chimeras that successfully transmitted the targeted allele through the germline. These chimeras gave rise to mice exhibiting consistent phenotypes. The resulting C57BL/6J Cd320^tm1Lcb mice, with both alleles flanked by loxP sites, were bred to homozygosity. Subsequently, these mice were crossed with the B6.FVB-Tg(EIIa-Cre)C5379Lmgd/J strain to induce Cre-mediated recombination. Following germline transmission of the deletion, the offspring were intercrossed, and those lacking the Cre transgene were selected, yielding the homozygous knockout strain Cd320^tm1.1Lcb. PCR-based genotyping was utilized to distinguish wild-type (WT), targeted, and deleted alleles.