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- 저자 : Kevin D. Wells (검색결과 2 건)
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Gene Editing of CD163 Protects Pigs from PRRSV Infectivity
Kristin M Whitworth,Kevin D. Wells,Randall S. Prather 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically important disease in swine in North America, Europe and Asia. PRRS is caused via infection of the pulmonary alveolar macrophages (PAMs) with the PRRS virus (PRRSV) causing respiratory illness and high fever in young growing pigs that predisposes them to secondary bacterial infections. PRRSV also causes severe reproductive failure in sows and boars. Although research is ongoing, PRRSV continues to elude a successful vaccine. In 2014, piglets were born with a gene edit in exon 7 of the Cluster of differentiation 163 (CD163) gene introduced by using the CRISPR/Cas9 site-directed nucleases system. The resulting litters of pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163-/- pigs were also challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the CD163+/- fetuses that have functional CD163 protein. The wild-type sow and fetuses were actively infected with the PRRSV and one sow aborted. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure. The results of this study clearly demonstrate that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. This is a great example of the potential of utilizing gene editing to improve animal agriculture.
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Beaton, Benjamin P,Kwon, Deug-Nam,Choi, Yun-Jung,Kim, Jae-Hwan,Samuel, Melissa S,Benne, Joshua A,Wells, Kevin D,Lee, Kiho,Kim, Jin-Hoi,Prather, Randall S John WileySons, Ltd 2015 Xenotransplantation Vol.22 No.5
<P><B>Background</B></P><P>Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (<I>CMAH</I>) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (<I>GGTA1</I>) in the <I>CMAH</I> KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting.</P><P><B>Methods</B></P><P>Zinc-finger nucleases from a previous <I>CMAH</I> KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified <I>GGTA1</I> cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in <I>CMAH</I> and <I>GGTA1</I> for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in <I>CMAH</I> and <I>GGTA1</I> double KO pig cells and were compared to wild-type and <I>CMAH</I> KO cells.</P><P><B>Results</B></P><P>Introduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of <I>CMAH</I>; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the <I>GGTA1</I> KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of <I>GGTA1</I>. As the length of donor DNA increased, the bi-allelic disruption of <I>GGTA1</I> increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of <I>GGTA1</I> expression and the decrease in gene expression sialyltransferase-related genes.</P><P><B>Conclusions</B></P><P>The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the <I>CMAH</I>/<I>GGTA1</I> double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.</P>
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