Porcine model of hemophilia A.

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate

Intraoperative laparoscopic detection of sentinel lymph nodes with indocyanine green and superparamagnetic iron oxide in a swine gallbladder cancer model.

Mapping of sentinel lymph nodes (SLNs) can enable less invasive surgery. However, mapping is challenging for cancers of difficult-to-access visceral organs, such as the gallbladder, because the standard method using radioisotopes (RIs) requires preoperative tracer injection. Indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) have also been used as alternative tracers. In this study, we modified a previously reported magnetic probe for laparoscopic use and evaluated the feasibility of detecting SLNs of the gallbladder using a laparoscopic dual tracer method by injecting ICG and SPIO into five swine and one cancer-bearing swine. The laparoscopic probe identified SPIO nanoparticles in the nodes of 4/5 swine in situ, the magnetic field counts were 2.5-15.9 μT, and fluorescence was detected in SLNs in all five swine. ICG showed a visual lymph flow map, and SPIO more accurately identified each SLN with a measurable magnetic field quite similar to the RI. We then developed an advanced gallbladder cancer model with lymph node metastasis using recombination activating gene 2-knockout swine. We identified an SLN in the laparoscopic investigation, and the magnetic field count was 3.5 μT. The SLN was histologically determined to be one of the two metastatic lymph nodes. In conclusion, detecting the SLNs of gallbladder cancer in situ using a dual tracer laparoscopic technique with ICG and SPIO was feasible in a swine model

<i>F8</i> targeting and genetic analysis of the colony 134-derived fetus.

<p>PCR analysis of genomic DNA of 134-fetus was shown. (<b>A</b>) Two or three independent PCR reactions were carried out for detection of recombination in <i>F8</i> of 134-fetus. (<b>B</b>) Southern blotting with a 5′ exon 14 probe (on <i>Sac</i> I− or <i>Sac</i> I + <i>Stu</i> I-digested DNA) and with a 3′ exon 22 probe (on <i>Sph</i> I− or <i>Xba</i> I-digested DNA) showed correct targeting of the <i>F8</i> in 134-fetus.</p

Analysis of the <i>F8</i> in cloned piglets.

<p>(<b>A</b>) PCR analysis of genomic DNA of piglet DNA was shown. Genomic DNA of wild-type, 134-fetus, piglet #1, piglet #2, piglet #3, and piglet #4 was subjected to PCR analysis with primers Exon 14 sF and Exon 18 sR as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049450#pone-0049450-g001" target="_blank">Figure 1</a>. The 8.3 kb exon 14–18 band was amplified from the 134-fetus DNA and the cloned piglet DNA. (<b>B</b>) Southern blotting with a 5′ exon 14 probe (on <i>Sac</i> I− or <i>Sac</i> I + <i>Stu</i> I-digested DNA) showed the same mobility shifts of the bands as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049450#pone-0049450-g002" target="_blank">Figure 2<b>B</b></a> and confirmed the insertion of the Neo resistant gene in <i>F8</i> of the cloned piglets. (<b>C</b>) RT-PCR analysis of piglet liver RNA was shown. Two independent PCRs (exons 14–16 and exons 18–22) revealed the absence of FVIII mRNA from the liver of cloned piglet #3. Control GAPDH mRNA was detected in the liver RNA of piglet #3 as in the wild type (WT).</p

The bleeding phenotype of cloned <i>F8</i> KO piglets.

<p>(<b>A</b>) A part of macroscopic picture of cloned piglet #1, which died by day 2 after birth is shown. Ecchymosis was seen in the cheek, the forelimb, and the hind limb (not shown). Pathological examination revealed hematomas in these areas of piglet #1. (<b>B</b>) Forelimb of cloned piglet #4 on day 1 after delivery was shown. Ecchymosis had been seen in the left forelimb of cloned piglet #4 since delivery. (<b>C</b>) On day 5 after administration of human FVIII (150 U/kg), the bleeding in the left forelimb was not observed. Macroscopic picture of cloned piglet #4 on day 28 after birth showed that the left forelimb was swollen because of the repeated bleeding (<b>D</b>), causing the piglet to limp (also see video 1).</p