Murine Surf4 is essential for early embryonic development

Abstract

Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.

Fig 1. Generation of Surf4 mutant alleles.

(A) Surf4 gene structure. Exons are shaded light blue for untranslated regions or dark blue for coding sequence. The target site for the sgRNA used for oocyte editing is indicated by the black triangle. (B) Mouse ES cells were either untreated or electroporated with plasmids for CRISPR/Cas9 disruption of the Surf4 target site. PCR amplification of genomic DNA or water control across the Surf4 target site revealed higher and lower molecular weight DNA fragments suggestive of nonhomologous endjoining repair of Surf4 indels. (C) The major PCR product was gel purified and subjected to T7 endonuclease I digestion. T7E1 digestion produced novel DNA fragments (arrows) indicating the presence of insertions/deletions in Surf4 exon 2. Wild type DNA was resistant to T7E1 digestion. (D) Sanger sequencing chromatograms of Surf4 target site amplicons of progeny from matings between Surf4-targeted founder mice and wild-type C57BL6/J mice. (E) DNA and predicted protein sequences for the 4 individual allele generated by CRISPR/Cas9 gene-editing of Surf4.

Fig 2. Surf4^+/- mice exhibit partial reduction of Surf4 transcripts with preferential loss of the mutant allele.

Sanger sequencing of the Surf4 target site was performed on PCR amplicons derived from genomic DNA (A) or reverse-transcribed cDNA (B) prepared from liver tissue of 3 Surf4^+/- mice. Decomposition of chromatograms was performed to quantify the relative proportion of each allele in each sample. (C) Total Surf4 transcript levels in liver tissue from 4 Surf4^+/+ and 4 Surf4^+/- mice were quantified and normalized to a panel of housekeeping genes by qRT-PCR.

Fig 3. Surf4 haploinsufficiency does not affect baseline plasma levels for PCSK9, ApoB, or cholesterol levels.

Plasma samples collected from 10 Surf4^+/- mice (heterozygous for the del(1) allele) and 6 wild-type littermate controls were assayed for plasma levels of total cholesterol (A), PCSK9 (B), and ApoB (C). Values were measured and averaged for each of two independent phlebotomies from each mouse. Both male and female mice were tested for each genotype. Significance testing was calculated by Student’s t-test between genotype groups.

Fig 4. Surf4 haploinsufficiency causes hepatic accumulation of apolipoprotein B but not PCSK9.

Liver lysates from 3 male Surf4^+/- mice harboring the del(1) allele and 3 male Surf4^+/+ littermate controls were immunoblotted for PCSK9, LDL receptor, apolipoprotein B, and alpha-tubulin. Densitometry values for PCSK9, LDLR, and apolipoprotein B were normalized to alpha-tubulin. Significance testing was performed by Student’s t-test between genotype groups.