Generation of Ptdsr-deficient mice
To investigate in vivo the functions of the phosphatidylserine receptor Ptdsr, we generated a null allele in the mouse by gene targeting (Figure 1a,1b,1c). In contrast to previously described Ptdsr-knockout mice [31,32], we used Bruce4 embryonic stem (ES) cells for gene targeting [33], thus generating a Ptdsr-null allele in a pure, isogenic C57BL/6J genetic background. The newly established knockout mouse line was named Ptdsrtm1Gbf (hereafter referred to as Ptdsr -/-). Heterozygous Ptdsr+/- mice were viable and fertile and showed no obvious abnormalities. Ptdsr +/- mice were intercrossed to generate homozygous Ptdsr-deficient mice. The absence of Ptdsr expression in Ptdsr -/- embryos was confirmed by RT-PCR (data not shown), and by northern and western blotting analyses (Figure 1d,e). Interbreeding of heterozygous mice showed that the mutation was lethal, since homozygous mutants were not detected in over 100 analyzed litters at weaning. To determine the stages of embryonic development affected by the Ptdsrtm1Gbf mutation, timed breedings were followed by PCR genotyping (Figure 1c) of embryos. We recovered fewer than the expected number of homozygous embryos from intercrosses of Ptdsr+/-mice. From a total of 1,031 embryos analyzed between gestational day (E) 9.5 and E18.5, 198 (19.2%) Ptdsr-deficient homozygous embryos were harvested, indicating that the introduced mutation is associated with a low rate of embryonic lethality in utero.
From E9.5 to E12.5, Ptdsr -/-  embryos were viable and of normal size. At E13.5 and thereafter, however, most Ptdsr -/-embryos showed morphological abnormalities (Table 1). All homozygous embryos harvested were growth-retarded from E13.5 onwards, had a pale appearance, and displayed multiple developmental dysmorphologies. These included various head and craniofacial malformations, such as exencephaly, cleft palate and abnormal head shape (Figure 1f,g). Gross inspection revealed that eye development was severely affected in 14.1% of homozygous embryos. The affected animals displayed a complete unilateral or bilateral absence of the eyes (Table 1) that was never detected in Ptdsr +/+ or Ptdsr +/- littermates. Furthermore, homozygous embryos harvested between E12.5 and E15.5 had subcutaneous edema (Figure 1f,g). Because we were able to recover Ptdsr -/- embryos until E18.5, we investigated whether Ptdsr-knockout mice could be born alive. Careful observation of timed matings allowed us to recover Ptdsr -/- neonates, but homozygous pups died during delivery or within minutes after birth. Ptdsr-deficient neonates were also growth-retarded, had a pale appearance and displayed various malformations. These included cleft palate, abnormal head shape, absence of eyes and edematous skin (Figure 1h). Thus, deletion of the Ptdsr gene resulted in perinatal lethality with variable severity and penetrance of phenotypes.