Lab Anim Res.  2018 Dec;34(4):279-287. 10.5625/lar.2018.34.4.279.

CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model

• Lee H
• Kim JI
• Park JS
• Roh JI
• Lee J
• Kang BC
• Lee HW

• KMID: 2459306
• DOI: http://doi.org/10.5625/lar.2018.34.4.279

Abstract

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

Keyword

PLAC8; CRISPR/Cas9; mouse model

MeSH Terms

Adipose Tissue
Animals
Body Weight
Codon, Initiator
DNA
Exons
Humans
Intestines
Lung
Male
Mice
Mice, Knockout*
Neomycin
Obesity
Placenta
Polymerase Chain Reaction
RNA, Guide
RNA, Messenger
Spleen
Zygote
Codon, Initiator
DNA
Neomycin
RNA, Guide
RNA, Messenger

Figure

• 

  Figure 1 CRISPR/Cas9-mediated generation of a Plac8 KO mouse model. (A) A representative view of the CRISPR/Cas9 targeting strategy used for generating Plac8 knockout (KO) mice. The nucleotide and amino acid (in purple) sequences of wild-type (WT) and KO alleles are shown. Exons are indicated with black and gray boxes, and introns are noted by black lines. The targeting sites are indicated by yellow arrows, and the binding sites for single guide RNAs (sgRNAs) are marked in blue, with the protospacer adjacent motif (PAM) sequence in red. The conventional start codon (ATG) is indicated in green. (B) A representative PCR genotyping result for Plac8 WT, homozygous (KO), and heterozygous (Het) KO mice. (C) Relative Plac8 mRNA levels in the intestine, liver, heart, lung, and brown adipose tissue (BAT) of the Plac8 KO (Black bar) mice were compared to WT (White bar). The mRNA levels were normalized by Gapdh. Graph shows mean±standard deviation.

• 

  Figure 2 Measurement of physiological parameters in Plac8 KO mice and comparison to WT littermate controls. (A and B) Body weight changes in Plac8 WT and KO of male (A) and female (B) mice from 4 to 52 weeks of age. (C–F) The amount of daily food (C and D) and water (E and F) consumed by WT and Plac8 KO male (C and E) and female (D and F) mice from 4 to 52 weeks of age. WT animals are shown in white dots, and KO mice are shown in black dots. Male mice are indicated with squares (A, C, and E), and female mice are indicated with circles (B, D, and F). Graphs show mean± standard deviation. *P<0.05.

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