Generation of Conditional Knockout Alleles for PRUNE-1

Abstract

PRUNE1 is a member of the aspartic acid-histidine-histidine (DHH) protein superfamily, which could display an exopolyphosphatase activity and interact with multiple cellular proteins involved in the cytoskeletal rearrangement. It is widely expressed during embryonic development and is essential for embryogenesis. PRUNE1 could also be critical for postnatal development of the nervous system as it was found to be mutated in patients with microcephaly, brain malformations, and neurodegeneration. To determine the cellular function of PRUNE1 during development and in disease, we have generated conditional mouse alleles of the Prune1 in which loxP sites flank exon 6. Crossing these alleles with a ubiquitous Cre transgenic line resulted in a complete loss of PRUNE1 expression and embryonic defects identical to those previously described for Prune1 null embryos. In addition, breeding these alleles with a Purkinje cell-specific Cre line (Pcp2-Cre) resulted in the loss of Purkinje cells similar to that observed in patients carrying a mutation with loss of PRUNE1 function. Therefore, the Prune1 conditional mouse alleles generated in this study provide important genetic tools not only for dissecting the spatial and temporal roles of PRUNE1 during development but also for understanding the pathogenic role of PRUNE1 dysfunction in neurodegenerative or neurodevelopmental disease. In addition, from this work, we have described an approach that allows one to efficiently generate conditional mouse alleles based on mouse zygote electroporation.

Keywords: CRISPR/Cas9; Cre recombinase; PRUNE1; cerebellar Purkinje cells; conditional null allele; in vitro fertilization; neurodegeneration; zygote electroporation.

Figure 1

Generation of the Prune1 conditional allele by CRISPR/Cas9-based gene-targeting. (a) Genomic structure of the mouse Prune1 locus. The regions used for inserting two loxP sites that flank exon 6 (E6) are shown. (b) Schematic representation of a targeting procedure that allowed to efficiently generate the Prune1 conditional alleles within five months. It consists of three steps: (1) In vitro analysis to test targeting efficiency, (2) the first round of targeting to generate mice homozygous for 3′ loxP, and (3) the second round of targeting of the 5′ loxP in zygotes made from the sperm of mice homozygous for 3′ loxP. (c) PCR genotyping of mouse offspring generated from the second round of targeting. Among seven offspring, three contained homozygous 5′ loxP and heterozygous 3′ loxP (1, 6, and 7 indicated by arrows), which was further confirmed by DNA sequencing. The PCR products of mutant (mut) and wt, as well as the formation of heteroduplexes (which reflects heterozygosity), are indicated. Controls used in this genotyping were genomic DNA prepared from: (1) wt C57BL/6J; (2) and (3): Blastocysts carrying homozygous 3′ loxP, and (4) blastocyst carrying homozygous 5′ loxP. The base pair (bp) of DNA marker (M) is indicated.

Figure 2

Characterization of the Prune1^{Δexon6/Δexon6} as a null allele. (a) Western blot analysis, demonstrating the complete absence of PRUNE1 protein in mouse embryonic fibroblast (MEF) cells derived from E10.5 Prune1^{Δexon6/Δexon6} embryo. Lane 1, Protein molecular weight marker; Lane 2, wt MEF; Lane 3, the Prune1^{Δexon6/Δexon6} MEF. The arrow indicates PRUNE1 protein (~58 kDa) which was only detected in wt MEF cells. Ponceau S staining is shown as a normalization control. (b,c) Whole mount imaging of yolk sac and embryos dissected from Prune1^Δexon6 intercross at E10.5. The Prune1^{Δexon6/Δexon6} displayed an avascular yolk sac with primitive vascular plexus and pericardial effusion (arrowhead) compared to the control littermate. (d–g) Hematoxylin-eosin (H&E) staining of sections prepared from E10.5 yolk sac and embryos showing distended capillary with defective hematopoiesis (arrow in (e)) and a thin heart wall with severe reduction of cardiomyocytes and very little trabeculae (arrow in (g)) in the Prune1^{Δexon6/Δexon6} mutants compared to the control. (h,i) Immunostaining of transverse sections of E10.5 embryos with anti-Endomucin antibody (a marker of endothelial cells), demonstrating lack of invaded vascular plexus in the neural tube of Prune1^{Δexon6/Δexon6} compared to the control. Arrow indicates invaded blood vessel in the control neural tube.

Figure 3

Additional developmental defects presented in the Prune1^{Δexon6/Δexon6} mutants. (a,b) Wheat germ agglutinin (WGA) staining of transverse sections of E10.5 embryos showing significantly decreased hematopoietic cells in the aorta-gonad-mesonephros (AGM) of Prune1^{Δexon6/Δexon6} (arrow in (b)) compared to the control. (c,d) H&E staining of E10.5 placenta, indicating a significantly thinner layer of trophoblast cells in the chorionic labyrinth (marked by brackets) in the Prune1^{Δexon6/Δexon6} placenta compared to the wt control. (e,f) Immunostaining of E10.5 placenta with anti-E-cadherin antibody (a global marker of syncytiotrophoblast), demonstrating significantly decreased labyrinthine syncytiotrophoblasts (marked by brackets) in the Prune1^{Δexon6/Δexon6} placenta compared to the wt control.

Figure 4

Characterization of the Prune1^F/F/Pcp2-Cre mice with conditional knockout of PRUNE1 in cerebellar Purkinje cells. (a,b) Immunostaining with anti-Calbindin antibody (a marker for Purkinje cell), demonstrating loss of more than 90% of Purkinje cells in two months old Prune1^F/F/Pcp2-Cre cerebella. Lobe 1 of the cerebellum is indicated. (c,d) H&E staining further confirming the loss of Purkinje cells in two months old Prune1^F/F/Pcp2-Cre cerebella. Arrow indicates Purkinje cell layer, which was present in the control, but not in the Prune1^F/F/Pcp2-Cre cerebella.