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.

Development and calcium level changes in pre-implantation porcine nuclear transfer embryos activated with 6-DMAP after fusion

Im, Gi-Sun,Samuel, Melissa,Lai, Liangxue,Hao, Yanhong,Prather, Randall S. JOHN WILEY & SONS LTD 2007 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.74 No.9

<P>This study investigated the effect of treatment with 6-dimethylaminopurine (6-DMAP) following fusion on in vitro development of porcine nuclear transfer (NT) embryos. Frozen thawed ear skin cells were transferred into the perivitelline space of enucleated oocytes. Reconstructed oocytes were fused and activated with electric pulse in 0.3 M mannitol supplemented with either 0.1 or 1.0 mM CaCl<SUB>2</SUB>. In each calcium concentration, activated oocytes were divided into three groups. Two groups of them were exposed to either ionomycin (I + 6-DMAP or 6-DMAP alone. In experiment 2, fused NT embryos in 0.3 M mannitol containing 1.0 mM CaCl<SUB>2</SUB> were exposed to 6-DMAP either immediately or 20 min after fusion/activation. For 0.1 mM CaCl<SUB>2</SUB>, oocytes activated with either I + 6-DMAP or 6-DMAP alone showed a higher (P < 0.05) developmental rate to the blastocyst stage than those activated with an electric pulse alone (26.7 and 22.5 vs. 12.5%). For 1.0 mM CaCl<SUB>2</SUB>, oocytes activated with either I + 6-DMAP or 6-DMAP alone showed significantly higher (P < 0.05) developmental rate to the blastocyst stage (35.6 and 28.3 vs. 19.8%). Developmental rate to the blastocyst stage was (P < 0.05) increased in NT embryos activated with 6-DMAP 20 min after fusion. 6-DMAP made a higher and wider Ca<SUP>2+</SUP> transient compared to that induced by electric pulses (Fig. 3). The fluctuation lasted during the time that oocytes were cultured in 6-DMAP. Regardless of Ca<SUP>2+</SUP> concentration in fusion medium, activation with 6-DMAP following electric pulses supported more development of porcine NT embryos. Activation of NT embryos with 6-DMAP after fusion in the presence of 1.0 mM CaCl<SUB>2</SUB> could support better developmental rate to the blastocyst stage. Mol. Reprod. Dev. 74: 1158–1164, 2007. © 2007 Wiley-Liss, Inc.</P>

Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer

Im, Gi-Sun,Yang, Boh-Suk,Lai, Liangxue,Liu, Zhonghua,Hao, Yanhong,Prather, Randall S. JOHN WILEY & SONS LTD 2005 Molecular Reproduction and Development Vol.71 No.2

<P>Fragmentation occurs during early developmental stages of electrically activated oocytes and nuclear transfer (NT) embryos. It might contribute to the low developmental rate of porcine NT embryos. The present study was conducted to investigate whether the addition of sugars such as sorbitol or sucrose suppresses fragmentation and supports the development of electrically activated oocytes and NT embryos. The activated oocytes were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with sorbitol or sucrose for 2 days after electric activation, and then cultured in the PZM-3 for the remaining 4 days. The osmolarities of PZM-3, PZM-3 supplemented with 0.05 or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose were 269 ± 6.31, 316 ± 3.13, 362 ± 4.37, and 315 ± 5.03 mOsm, respectively. When parthenogentically activated oocytes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate were observed. Interestingly, addition of sugar to PZM-3 for 2 days reduced the fragmentation rate compared to PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 increased the fusion rate (84.2% ± 6.07 vs. 95.1% ± 2.52), cleavage rate (67.6% ± 5.80 vs. 77.3% ± 3.03), and developmental rate to the blastocyst stage (10.2% ± 0.79 vs. 19.4% ± 1.77). There was no significant difference between treatments for the number of the blastocysts. In addition the fragmentation rate was reduced compared to PZM-3 without sorbitol (26.1 ± 4.30 vs. 14.5 ± 1.74). In conclusion, increasing the osmolarity of PZM-3 through addition of either sorbitol or sucrose for 48 hr increased the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate through increasing osmolarity. Mol. Reprod. Dev. 71: 159–165, 2005. © 2005 Wiley-Liss, Inc.</P>

Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells

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>

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals

Im, Gi-Sun,Seo, Jin-Sung,Hwang, In-Sun,Kim, Dong-Hoon,Kim, Se-Woong,Yang, Byoung-Chul,Yang, Boh-Suk,Lai, Liangxue,Prather, Randall S. Wiley Subscription Services, Inc., A Wiley Company 2006 Molecular reproduction and development Vol.73 No.9

<P>Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%–26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%–23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos. Mol. Reprod. Dev. 1094–1101, 2006. © 2006 Wiley-Liss, Inc.</P>

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

Wu, Alexander T. H.,Sutovsky, Peter,Manandhar, Gaurishankar,Xu, Wei,Katayama, Mika,Day, Billy N.,Park, Kwang-Wook,Yi, Young-Joo,Xi, Yan Wei,Prather, Randall S.,Oko, Richard American Society for Biochemistry and Molecular Bi 2007 The Journal of biological chemistry Vol.282 No.16

Developmental competence of porcine parthenogenetic embryos relative to embryonic chromosomal abnormalities

Hao, Yan-Hong,Lai, Liang-Xue,Liu, Zhong-Hua,Im, Gi-Sun,Wax, David,Samuel, Melissa,Murphy, Clifton N.,Sutovsky, Peter,Prather, Randall S. Wiley Subscription Services, Inc., A Wiley Company 2006 Molecular reproduction and development Vol.73 No.1

<P>Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM<SUB>3</SUB>) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 ± 7.4 vs. 28.7 ± 3.7; 10.9 ± 2.8 vs. 21.5 ± 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 ± 1.8 vs. 29.7 ± 0.8; 29.7 ± 3.3 vs. 32.0 ± 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 ± 12.8 vs. 16.4 ± 7.4 on day 6), and increased with culture time (71.9 ± 12.1 vs. 10.9 ± 2.8. on day 7,and 75.0 ± 22.6 vs. 12.1 ± 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20–39, >40) and into three groups according to the morphological diameter (<150, 150–180, >180 µm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 µm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc.</P>

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

Alexander, T.H.Wu,Sutovsky, Peter,Manandhar, Gaurishankar,Xu, Wei,Katayama, Mika,N. Day, Billy,Park, Kwang-Wook,Yi, Young-Joo,Xi, Yan Wei,S.Prather, Randall,Oko, Rechard 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10