PRRSV infection of ΔSRCR5 pulmonary alveolar macrophages (PAMs) is halted prior to the formation of the replication/transcription complex.

<p>PAMs from wild type (top two panels), heterozygous (middle two panels), and ΔSRCR5 (bottom two panels) animals were inoculated at MOI = 2 with PRRSV genotype 1, subtype 3 (strain SU1-Bel). 22 hpi cells were fixed and stained with an anti PRRSV-nsp2 antibody, DAPI, and phalloidin. Scale bar represents 40 μm.</p

ΔSRCR5 pulmonary alveolar macrophages (PAMs) are not susceptible to infection with PRRSV genotype 1.

<p><b>A-C)</b> PAMs from wild type (red), heterozygous (blue), and ΔSRCR5 (green) animals were inoculated at MOI = 1 of PRRSV genotype 1, subtype 1 (strain H2, A), subtype 2 (strain DAI, B), and subtype 3 (strain SU1-Bel, C). 19 hpi cells were detached, fixed and stained with an anti PRRSV-N protein antibody and CD163. Infection was quantified by FACS analysis. Over 98% of infected macrophages were qualified as CD163 positive. Infection levels were statistically analyzed using an unpaired t-test of all wild type against all heterozygous or all biallelic / homozygous data points. Error bars represent SEM, n = 3. <b>D-F)</b> Replication growth curves of PRRSV genotype 1, subtype 1 (strain H2, C), subtype 2 (strain DAI, D), and subtype 3 (strain SU1-Bel, F). PAMs from wild type (red, 628 filled circle, 633 open circle), heterozygous (blue, 627 filled square, 633 open square), and ΔSRCR5 (green, 629 triangle pointing down, 630 triangle pointing up) animals were inoculated at MOI = 0.1 of the respective strain. Cell supernatant was collected at indicated time points to measure the released viral RNA by RT-qPCR. Error bars represent SEM, n = 3*2. <b>G-J)</b> Quantification of infectious particles produced at 48 hpi by TCID₅₀ analysis. Cell supernatant collected at the 48 hpi time point of infection of the time-course experiment was analyzed for infectious viral particle production quantified by TCID₅₀. Infection levels were statistically analyzed using an unpaired t-test of all wt against all het or all ΔSRCR5. Error bars represent SEM, n = 3.</p

ΔSRCR5 peripheral blood monocyte-derived macrophages (PMMs) are not susceptible to infection with PRRSV genotype 2.

<p>Replication of PRRSV on PMMs in long-term infections with genotype 2, <b>A)</b> highly virulent strain VR-2385, and <b>B</b>) highly pathogenic strain MN-184. PMMs from wild type (red, 628 filled circle, 633 open circle), heterozygous (blue, 627 filled square, 633 open square), and ΔSRCR5 (green, 629 triangle pointing down, 630 triangle pointing up) animals were inoculated at MOI = 0.1 of the respective strain. Cell supernatant was collected at indicated time points to measure the released viral RNA by RT-qPCR. Error bars represent SEM, n = 3*2.</p

Generation of an exon 7 deletion in CD163 using CRISPR/Cas9.

<p><b>A)</b> Schematic of the CD163 gene in the pig genome on chromosome 5. Indicated in red are the 16 exons encoding the CD163 mRNA, in varied colors underneath are the 9 scavenger receptor cysteine-rich (SRCR) domains that form the pearl on a string structure of the CD163 protein. Excision of exon 7 using two guide RNAs (sgSL26 & sgSL28) located in the flanking introns should result in SRCR 5 removal from the encoded protein. Indicated are also the locations of sgRNAs SL25 and SL27. <b>B)</b> <i>In vitro</i> assessment of guide RNAs sgSL25, sgSL26, sgSL27, and sgSL28. PK15 cells were transfected with either a single plasmid encoding a guide RNA + Cas9 or co-transfected with combination of two such plasmids. Transfected cells were identified by GFP expression and isolated by FACS. Cutting efficiency of single guide RNA transfection was assessed by a Cel1 surveyor assay. Relative efficiency of exon 7 deletion upon double transfection was assessed by PCR. <b>C)</b> Schematic of the Cas9/guide RNA injection into zygotes. The injection mix was injected into the cytoplasm of zygotes and contained uncapped, non-polyadenylated guide RNAs sgSL26 and sgSL28, as well as capped, polyadenylated Cas9 mRNA.</p

ΔSRCR peripheral blood monocyte-derived macrophages (PMMs) still function as hemoglobin-haptoglobin (Hb-Hp) scavengers.

<p><b>A)</b> Induction of HO-1 expression by Hb-Hp uptake. PMMs were incubated for 24 h in presence of 100 μg/ml Hb-Hp. RNA was isolated from cells and levels of heme oxygenase 1 (HO-1) mRNA determined by RT-qPCR (outlined bars uninduced, filled bars Hb-Hp uptake induced; red wild type, blue heterozygous, green ΔSRCR5). Expression levels were normalized using β-Actin expression levels and to the level of unstimulated HO-1 mRNA expression of each animal. Uninduced versus induced levels of HO-1 expression were analyzed by an unpaired t-test. Error bars represent SEM, n = 3. <b>B</b>) PMMs were incubated for 24 h in presence of 100 μg/mol Hb-Hp. PMMs were lysed with reducing SDS sample buffer and HO-1 protein expression analyzed by western blot. <b>C</b>) Quantification of Hb-Hp uptake. PMMs were incubated in presence of 10 μg/ml Hb_AF488-Hp for 30 min. Uptake of Hb_AF488-Hp was measured by FACS analysis (colored contour plots; red wild type, blue heterozygous, green ΔSRCR5) relative to isotype controls (grey). <b>D)</b> Visualization of Hb-Hp uptake. PMMs were incubated for 30 min with 10 μg/ml Hb_AF488-Hp. Cells were fixed, permeabilized and stained against CD163 and with DAPI. Representatively shown are pig 628 as wild type and 630 as ΔSRCR5 animals. Scale bar represents 40 μm.</p

Genotypes and growth of assessed F1 animals.

<p>Genotypes and growth of assessed F1 animals.</p

Immuno-histochemistry analysis of mammary tissue.

<p><b>Panel A:</b> Paraffin embedded sections of mammary tissue (day 10 lactation) were analysed using a rabbit-anti α-casein antiserum. The slides were subsequently counterstained with haematoxylin. Representative sections derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice are presented. The lower panels are control sections incubated with a rabbit pre-immune serum in place of the α-casein specific antiserum. <b>Panel B:</b> Representative sections derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice at day 10 of lactation stained with haematoxylin.</p

Comparison regarding general health and behaviour between pups nursed by wild-type dams and pups nursed by α-casein deficient dams.

<p>P-values of parameters without significant differences between group 1 (G1: wild-type pups nursed by wild-type dams) and 2 (G2: wild-type pups nursed by α-casein deficient dams; P-value G1 vs. G2) and group 1 and 3 (G3: heterozygous pups nursed by wild-type dams; P-value G1 vs. G3) at 8 weeks of age (Fisher's exact test and *Mann-Whitney-U test). Constant values indicate that all animals in the groups compared had the same, normal, score.</p

Impact of α-casein deficient milk on pup growth.

<p><b>Panel A:</b> Growth curve of three different groups of mice during lactation (G1: wild-type pups nursed by wild-type dams n = 34; G2: wild-type pups nursed by α-casein deficient [−/−] dams, n = 25; and G3: heterozygous pups nursed by wild-type dams, n = 22). Values shown are +/− standard deviation. All weight differences between group G2 vs. G1 and G3 were significant from day 7 (p<0.001) as assessed by ANOVA. <b>Panel B:</b> Percentage weight gain throughout different stages of life for the three experimental groups. The weight of individual mice was compared on two days (as indicated: e.g. 1/3 corresponds to the interval between day 3 and day 1 of life) and the percent weight increase was recorded. The average for all mice in the three experimental groups is shown for consecutive time periods. <b>Panel C:</b> Growth curve of mice in the three groups over the first 6 months of life. Mice nursed by α-casein deficient dams show a consistent growth deficiency. <b>Panel D:</b> Growth curve of mice in the three groups of mice over the first 6 months of life separated by gender. Error bars represent standard deviations.</p

Analysis of milk calcium and phosphate levels and milk protein gene expression.

<p><b>Panel A:</b> Calcium and phosphate content of mouse milk was determined as indicated in the methods section. Concentrations are given in nM. <b>Panel B:</b> Quantitative PCR analysis of α-casein and β-casein gene expression. cDNA derived from representative wild-type, heterozygous [+/−] and homozygous [−/−] α-casein deficient mice was analysed using primer pairs specific for α-casein, β-casein and the reference gene GAPDH. Expression of the casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel C:</b> Quantitative PCR analysis of γ-casein and κ-casein gene expression. Expression of the γ and κ-casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel D:</b> Correlation of casein gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Casein gene expression was correlated with the expression of the reference gene β-actin. Quantification of α-casein was done in 3 [+/+], 7 [+/−] and 5 [−/−] mice. Quantification of β-casein was done in 3 [+/+], 8 [+/−] and 4 [−/−] mice. Quantification of γ- and k-casein was done in 3 [+/+], 3 [+/−] and 3 [−/−] mice. Expression in heterozygous and α-casein deficient mice is presented as percentage of median casein gene expression in wild-type control mice [+/+] (set to 100%). Error bars represent standard deviations. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, p<0.01 by **, and p<0.001 by ***. Exact p values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021775#pone-0021775-t006" target="_blank">table 6</a>.</p