Selenoprotein I is essential for murine embryogenesis

Abstract

Selenoprotein I (SELENOI) is an ethanolamine phosphotransferase that catalyzes the third reaction of the Kennedy pathway for the synthesis of phosphatidylethanolamine. Since the role of SELENOI in murine embryogenesis has not been investigated, SELENOI^-/+ mating pairs were used to generate global KO offspring. Of 323 weanling pups, no homozygous KO genotypes were found. E6.5-E18.5 embryos (165 total) were genotyped, and only two E18.5 KO embryos were detected with no discernable anatomical defects. To screen embryos prior to uterine implantation that occurs ~ E6, blastocyst embryos (E3.5-E4.4) were flushed from uteruses of pregnant females and analyzed for morphology and genotype. KO embryos were detected in 5 of 6 pregnant females, and 7 of the 32 genotyped embryos were found to be SELENOI KO that exhibited no overt pathological features. Overall, these results demonstrate that, except for rare cases (2/490 = 0.4%), global SELENOI deletion leads to early embryonic lethality.

Keywords: Embryonic lethal; Phosphatidylethanolamine; Phospholipid transferase; Selenium.

Figure 1.

Strategy for generating and screening mice with SELENOI KO in all tissues. (A) A floxed mouse cell line was created on a C57BL/6 background using CRISPR/Cas9 to insert loxp sites flanking exons 2 and 3 in the SELENOI gene. PCR primer pairs were designed for genotyping the DNA of mouse tissues. (B) Results from one litter of E18.5 embryos show PCR results for primer pairs designed to detect the excised (top panel) and intact (bottom panel) SELENOI gene allele. (C) PCR results from the same litter show confirmation of intact SELENOI gene allele with two different primer pairs. (D) The combined PCR results allowed genotypes to be identified of all embryos from the E18.5 litter.

Figure 2.

The rare E18.5 SELENOI KO embryo does not exhibit any pathological features. Representative sagittal sections from all three genotypes of E18.5 from the same litter are shown. No distinct features were found in SELENOI^−/− embryos compared to SELENOI^−/− and SELENOI^−/− littermates.

Figure 3.

Genotyping blastocyst embryos prior to uterine implantation reveals the presence of SELENOI^−/− embryos. (A) Pregnant female mice were euthanized and ovaries/ovaducts/uteruses removed. Blastocyste embryos were flushed from ovaducts and visualized for stage of development (E3.5–4.5). The morphology of the blastocyst embryos at this ~100 cell stage for SELENOI KO did not show any pathological features. (B) Primers were designed for distinguishing between KO (SELENOI^−/−) and non-KO (SELENOI^−/+ or ^+/+) using PCR analyses of cDNA. (C) PCR results showed the presence of 1 KO embryo out of 8 embryos from one pregnant female. (D) Cumulative results from 6 pregnant females shows the presence of 7 KO blastocysts.