Figure 2. Construction of Plscr1 knockout mice. (A) The
schematic drawing of the strategy for Plscr1 gene disruption. The
target sites of clustered regularly interspaced short palindromic repeat
(CRISPR)-Cas9 small guide RNAs (sgRNAs) in the Plscr1 gene are
indicated in red, and the deleted region in the Plscr1 gene of
knockout mice is indicated by dashes. The positions of RT-PCR primers
are indicated by arrows. (B) The schematic drawing of the domain
structure of PLSCR1 in wildtype and knockout mice. (C)The expression ofPlscr1 mRNA in the tail of P42 mice was determined by RT-PCR.
β-actin was included as the internal control. (D) Western blot showed
the expression in the liver and lung of P42 mice. (E) PLSCR1 was
detected by immunohistology using rabbit anti-mPLSCR1, visualized with
3,3,-diaminobenzidine, and counterstained with hematoxylin. Scale bar:
20 μm. +/+:wild type mice; +/-: heterozygous mice; -/-:Plscr1knockout mice.
We then examined whether Plscr1 knockout affects H1N1 SIV
infection. Control and Plscr1 -/- mice were
infected intranasally with 103 pfu H1N1 SIV, and the
survival rate, body weight and lung virus load were determined over a
28-day period. The results show that the survival rate ofPlscr1 -/- mice is much lower than that of
control mice after SIV infection (Figure 3A).Plscr1-/- mice start to die at 3 dpi, and the
survival rate drops to 50% at 28 dpi. Meanwhile, similar body weight
was observed between survived Plscr1 -/- mice
and control mice (Figure 3B). Consistent with the decreased survival
rate, higher lung viral load was observed inPlscr1 -/- mice (Figure 3C). Titers of SIV
increase in the lungs of both genotypes and peak at 5 dpi, then start to
decrease and not detectable at 14 and 28 dpi. Virus titers are
significantly higher in Plscr1 -/- mice at all
time points examined (Figure 3C). Consistently, western blotting shows
that the level of NP in Plscr1-/- mice was
significantly higher than that in wild mice at 3 dpi (Figure 3D and 3E).
Taken together, our present data suggest that H1N1 SIV replication is
increased in Plscr1 knockout mice that leads to lower survival
rate.