Use of gene-editing technology to introduce targeted modifications in pigs

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

Gene expression of CCL8 and CXCL10 in peripheral blood leukocytes during early pregnancy in cows

Background: The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes(PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination(AI). Based on the day of return of estrus, cows were divided into three groups, pregnant(n = 5), early embryonic mortality(EEM; n = 5) and late embryonic mortality(LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.Results: The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d(P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 k Da(ISG15), myxovirus-resistance gene(MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows(P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ(IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT(100 ng/mL) and CCL16(100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA(P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16(P < 0.05).Conclusions: The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events.The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as wel as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.

SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9(H3K9)in the zygotic pronuclei improves porcine embryo development

Background: The histone code is an established epigenetic regulator of early embryonic development in mammals.The lysine residue K9 of histone H3(H3 K9) is a prime target of SIRT1, a member of NAD+-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3 K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3 K9 methylation and acetylation in porcine zygotes and the significance of H3 K9 modifications for early embryonic development.Results: Our results show that SIRT1 activators resveratrol and BML-278 increased H3 K9 methylation and suppressed H3 K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3 K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate(5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling.Conclusions: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3 K9 me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.

Low density lipoprotein in cryopreservation of semen

Artificial insemination made a predominant contribution towards the improvement of genetic potential and increased productivity in animal husbandry sectors. In frozen semen technology, about 50% of sperm died because of cryoinjury or cryodamage during the process of cryopreservation and thawing of the semen. These cryo-damages can be minimized by use of various cryoprotective agents or cold shock absorbers in the frozen semen technology. Of which, egg yolk (EY) is most primarily important in the extender preparation for various mammalian species for longer period and it works against the cold shock during the cryopreservation process due to the presence of low density lipoproteins (LDL). Further, EY contains substances other than LDL that affect the sperm quality parameters especially reduced motility and inhibit the respiration of sperm;therefore, there was heavy demand on replacement of the EY with the particular responsible substances (LDL) in the semen extender. Later on, various investigators tried to extract the LDL from the EY of hen and finally succeeded to extract the LDL from the EY of hen for semen preservation of various species. The concentration of LDL used in various species is varied and this may be due to the composition and concentration of phospholipids, cholesterol and its proportion in the sperm membrane. In bovine species, the concentration of LDL was standardized as 8% (w/v) on dry matter basis. This is equal to 20% EY used in conventional semen extender. As it is explained that the 20% EY contains 68% LDL (13.6 g) and on dry matter basis, it is approximately 60% (8.16 g). This calculation indicates 20% EY contains 8% LDL on dry matter basis. The LDL protects the sperm by various mechanisms to maintain the integrity of sperm membrane, which is explained in the present review. It was concluded that the investigation is still to be carried out to find out the exact roles of apoproteins and lipids of LDL and to indentify and isolate the detrimental substances presented in the whole EY.

Production of chicken chimeras by fusing blastodermal cells with electroporation

Aim: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cellsfused with electroporation. Methods: Stage-X blastodermal cells isolated from freshly laid fertile unincubated whiteLeghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferredto the recovery medium (DMEM containing 10% FBS) and was injected into the subgenninal cavity of recipient unin-cubated embryos (stage X). Results: Of 177 recipient embryos injected with the fusing blastodermal cells, 6(3.4 %) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence offeathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days ofincubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromo-some-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other ex-hibited the same DNA in the left and right gonads and other tissues, but not the stomach. Conclusion: Recipientembryo having electrofused blastodennal cells yields somatic and germline chimeric chickens more successfully.(Asian J Androl 2000 Dec; 2: 271-275)

Impact of Presence or Absence of Trehalose during Vitrification on Viability and Development of Vitrified/Warmed Immature Dromedary Camel Oocytes

Vitrification of immature oocytes at the germinal vesicle (GV) stage is important to preserve female gametes. The standard formula for vitrification solutions has long been a debate. Herein, we investigated the effect of the presence or absence of trehalose in vitrification solution on viability, in vitro maturation (IVM) rates, and development of vitrified/warmed immature dromedary camel oocytes. Cumulus oocyte complexes (COCs) obtained at slaughter from the ovaries of mature she-camels were randomly allocated into three groups;namely, control group, oocytes were directly subjected to IVM without vitrification, vitrification solution 1 (VS1) group, oocytes were vitrified in a solution composed of 25% ethylene glycol (EG) plus 25% dimethyl sulfoxide (DMSO) + 0.5 M trehalose;and vitrification solution 2 (VS2) group, oocytes were vitrified in a solution composed of 25% EG plus 25% DMSO. Vitrification of COCs was conducted by open pulled straws (OPS) method. Following vitrification and warming, morphologically viable oocytes were matured in vitro for 36 h. COCs were then fertilized and cultured in vitro for 7 days. The percentage of viable oocytes was significantly higher (P 0.05) in VS2 than VS1 group (80.0% vs. 63.3%, respectively). Nuclear maturation, cleavage (48 h post-insemination;pi), and blastocyst rates (7 days pi) were significantly higher (P < 0.05) in VS2 than in VS1 groups. No significant differences were observed in oocyte maturation and development rates between VS2 and control groups. In conclusion, vitrification of immature dromedary camel oocytes in trehalose-free solution (VS2) was more advantageous than that in trehalose supplemented media since it did not reduce viability and development.

A simplified protocol for the semi-large scale recovery of plasmids from <i>Escherichia coli</i>grown on agar plates

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells(HDDPCs) and HDDPC-derived iPS cells

The ability of human deciduous tooth dental pulp cells（HDDPCs） to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a Piggy Bac（PB）-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing td Tomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Osteopontin is a biomarker for early autoimmune uveoretinitis

Osteopontin(OPN)is an extracellular matrix protein with a diverse range of functions,including roles in cell adhesion,migration,and immunomodulation,which are associated with the modulation of neuroinflammation in the central nervous system.The present study was performed to evaluate the involvement of OPN in the eyes of an experimental autoimmune uveoretinitis(EAU)model.The EAU model was developed by immunization of Lewis rats with interphotoreceptor retinoid-binding protein.The results showed the OPN level was remarkably upregulated in the eye of EAU rats on day 9 post-immunization.The level of CD44,a ligand of OPN,was increased in the ciliary body of EAU rats.Furthermore,OPN was also detected in the ciliary body and activated microglia/macrophages in the EAU retina.The results suggest that OPN was significantly upregulated in the eyes of EAU rats,and that it may be useful as an early biomarker of ocular autoimmune diseases.All animal experiments were approved by the Institutional Animal Care and Use Committee of Jeju National University(approval No.2020-0012)on March 11,2020.

Evaluatingα-galactosylceramide as an adjuvant for live attenuated infuenza vaccines in pigs

Natural killer T(NKT)cells activated with the glycolipid ligandα-galactosylceramide(α-GalCer)stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses.Several studies have used this approach to adjuvant inactivated and subunit infuenza A virus(IAV)vaccines,including to enhance cross-protective infuenza immunity.However,less is known about whetherα-GalCer can enhance live attenuated infuenza virus(LAIV)vaccines,which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating infuenza vaccines.The current study used the swine infuenza challenge model to assess whetherα-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine(TX98ΔNS1)encoding a truncated NS1 protein.In one study,weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0,10,50,and 100μg/kg doses ofα-GalCer,and subsequently challenged with a heterologous H3N2 virus.All treatment groups were protected from infection.However,the addition ofα-GalCer appeared to suppress nasal shedding of the LAIV vaccine.In another experiment,pigs vaccinated with the H3N2 LAIV,with or without 50μg/kg ofα-GalCer,were challenged with the heterosubtypic pandemic H1N1 virus.Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways,and signifcantly decreased virus shedding.On the other hand,combining the vaccine withα-GalCer reduced cross-protective cellular and antibody responses,and resulted in higher virus titers in respiratory tissues.These fndings suggest that:(i)high doses ofα-GalCer impair the replication and nasal shedding of the LAIV vaccine;and(ii)α-GalCer might interfere with heterosubtypic cross-protective immune responses.This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines.

Production of germline chimeric quails following spermatogonial cell transplantation in busulfan-treated testis

Dear Editor, The merits of using quaff as an avian experimental model include high egg production, low maintenance cost, small body size, and short generation period （approximately 6-8 weeks）. Indeed, these characteristics make quail an ideal species for many biological areas including transgenic research. Methods of primordial germ cell （PGC）-mediated germline chimera production are considered very reliable and have been used in avian transgenesis.

Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

Negative biomarker-based male fertility evaluation： sperm phenotypes associated with molecular-level anomalies

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry （FC）. A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies （ART） and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA （Lens culinaris agglutinin; reveals altered sperm surface） and PNA （Arachis hypogaealpeanut agglutinin; reveals acrosomal malformation or damage）. The Postacrosomal Sheath WWl Domain Binding Protein （PAWP）, implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium

Cudrania tricuspidata Bureau(CTB),a species of the Moraceae plant,has been used as a bruise recovery treatment.This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease(IBD).We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C.CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB,which are responsible for the expression of cell-cycle-related proteins(CDK2,CDK4,cyclin D1 and cyclin E)during its promotion of cell proliferation.Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4%(W/V)for seven days.We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score,which was composed of body weight change,diarrhea,and hematochezia in ICR mice treated with dextran sulfate sodium.Hence,we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC,JNK and NF-κB in colon epithelial cells.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing

Dioscorea batatas Decne(DBD)has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia.As a source of therapeutic agents,many glycoproteins have been isolated from mushrooms and plants,but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet.In the present study,we investigated the wound healing potentials of the 30 kDa glycoprotein(DBD glycoprotein)isolated from DBD in human intestinal epithelial(INT-407)cells.We found that DBD glycoprotein(100μg·mL^-1)significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C(PKC).DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase(MAPK),which is responsible for the phosphorylation of NF-κB inhibitorα(IκBα).DBD glycoprotein increased the level of profilin-1(PFN1),α-actinin and F-actin expression via activation of transcription factor,nuclear factor-kappa B(NF-κB)during its promotion of cell migration.Experimental mouse colitis was induced by adding dextran sulfate sodium(DSS)to the drinking water at a concentration of 4%(W/V)for 7 days.We figured out that administration of DBD glycoprotein(10 and 20 mg·kg-1)lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice.In this regard,we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Supplemental iodine as a key to reproduction in pandas?

Pandas are endemic to iodine-poor environments and appear to be specialized for a goitrogenic staple diet.In particular,the importance of thiocyanate in bamboos might possibly have been overlooked in captive breeding programs.Although excreted in urine,thiocyanate first antagonizes absorption of iodine by the thyroid(of par­ent,fetus and suckling juveniles)and the mammary glands.In livestock and humans,subclinical deficiency of iodine is known to result in reproductive problems(including retardation of the fetus and suckling infant)even where the mother appears to be unaffected beyond slight hyperplasia of the thyroid and subtle hypothyroid­ism as reflected by levels of thyroid hormones.We suggest that the possibilities of iodine deficiency or excess should be carefully considered wherever the reproductive rates of pandas are unsatisfactory.

Re： Is PAWP the ＇real＇ sperm factor？

This letter is in response to an article written by Michail Nomikos, Karl Swann and F. Anthony Lai in the Research Highlights section of the Asian Journal of Andrology （AJA）. The article is entitled, ＂Is PAWP the ＇real＇ sperm factor？＂ and was written in response to our article entitled, ＂Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in human and mouse＂ published recently in FASEB Jol According to the Science Editor of AJA, ＂Research Highlight＂ pieces are brief articles that are meant to report on publications from the primary literature. Along those lines, we were delighted to read an insightful comment on our FASEB article prepared for AJA by Dr. George L Gerton.