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Alpha-1, 3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity.

INTRODUCTION

Despite an increased demand of human organs for transplantation, supply of transplantable human organs is limited. Xenotransplantation may solve this shortage of organs. Among several species, pigs are considered as the most probable species for xenotransplantation because of their compatible size and physiological similarities to humans, breeding characteristics, and availability in specific pathogen-free (SPF) conditions [1,2]. However, porcine organs transplanted to humans may cause severe immune rejection [3]. Hyperacute rejection (HAR) that occurs immediately after transplantation is a major hurdle in pig-to-human transplantation [4,5]. The presence of the epitope galactose-alpha-1,3-galactose ([alpha]Gal) synthesized by alpha-1,3-galactosyltransferase ([alpha]GT) on the surface of porcine cells induces HAR [4,5]. Therefore, the production of [alpha]GT-deficient pigs is essential to avoid HAR in pig-to-human xenotransplantation [6].

In an effort to overcome HAR, pigs with targeted disruption of the [alpha]GT gene have been produced by somatic cell nuclear transfer (SCNT) [7-10]. Even after the production of animals with heterozygous disruption of the gene, the procedure to produce homozygote pigs requires a great deal of cost, time, and labor. Heterozygote pigs should be either bred at least for two generations to ultimately obtain homozygote pigs.

The procedure to isolate homozygous [alpha]GT knockout (KO) cells derived from loss of heterozygosity (LOH) in heterozygous KO cells has been developed [11,12]. Biotin-labeled IB4-lectin and streptavidin-conjugated beads were used to remove cells expressing [alpha]Gal on their surface. Using this method and subsequent nuclear transfer (NT), homozygous [alpha]GT KO pigs were produced from fetal fibroblasts [11]. However, the procedure involves sacrificing heterozygous animals due to collection of fetuses before their term, and it has not been determined whether such procedure could be applied to postnatal cells obtained from heterozygous [alpha]GT KO pigs. It would be particularly beneficial for animals with long gestation period to save the time required for breeding for the production of homozygote animals from the verified heterozygotes.

The aim of the current study is to isolate homozygous [alpha]GT KO cells from postnatal heterozygous [alpha]GT KO skin fibroblasts and ultimately to evaluate the developmental competence of isolated homozygote cells after SCNT.

MATERIALS AND METHODS

Animal ethics

All procedures in this study were carried out in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Institutional Animal Care and Use Committee, Dankook University.

Chemicals and reagents

Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of porcine fetal fibroblasts

Pig fetuses at 28 to 39 days of gestation were obtained from Minnesota miniature pigs maintained in SPF conditions at Seoul National University. The head, dorsal spine of the medial section, and tail were removed before collection of embryonic fibroblasts. Briefly, small pieces of remaining tissues were washed in Dulbecco's phosphate-buffered saline (DPBS; Invitrogen, Carlsbad, CA, USA) and minced with a surgical blade in a 100-mm petridish. Cells were then dissociated from the tissues in 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Invitrogen, USA) for 10 min at 39[degrees]C. After centrifuging the cell suspension three times at 800xg for 5 min, pellets were resuspended and seeded on 100-mm culture dishes (Corning, Glendale, AZ, USA) and cultured for 6 to 8 days in Dulbecco's modified Eagle medium (DMEM; Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 1 mM L-glutamine, 100 units/mL penicillin, and 0.5 mg/mL streptomycin in a humidified atmosphere of 5% C[O.sub.2] in 95% air. After removal of unattached clumps of cells by washing culture plates with DMEM, attached cells were further cultured until confluent and subcultured at intervals of 5 to 7 days by trypsinization until used for transfection and SCNT.

Transgenesis of porcine fetal fibroblasts

Fetal fibroblasts were cultured to 80% to 90% confluence on 150-mm plates. After harvested with trypsin and washed with DPBS, cells were transfected using Gene Pulser II (Bio-Rad Laboratories, Hercules, CA, USA). Gene targeting vector, as shown in Figure 1, was introduced into fetal fibroblasts. A total of 2x[10.sup.7] cells were suspended in 400 [micro]L of transfection buffer with 10 [micro]g of linearized targeting vector. Electroporation was performed at 250 V, 1,000 mF. Starting from 24 h after transfection, genetically modified cells were selected with culture medium containing 500 [micro]g/mL of G418 (Invitrogen, USA) for 10 to 14 days. After antibiotic selection, G418-resistant colonies were isolated and transferred onto 0.1% gelatin-coated dish for clonal culture. Cells were continuously cultured at 39[degrees]C in a humidified atmosphere containing 5% C[O.sub.2] and 95% air. Genomic DNA was extracted from cells of G418-resistent colonies, and [alpha]GT gene targeting events were screened by polymerase chain reaction (PCR) using specific primer sets described previously [2].

Nuclear transfer and embryo transfer

The in vitro maturation (IVM) of oocytes and NT were performed as described previously [2]. Briefly, 42 h after the onset of IVM, oocytes were enucleated with a 20-[micro]m (internal diameter) glass pipette by aspiration of the first polar body and the second metaphase plate with a small volume of surrounding cytoplasm in HEPES-buffered TCM-199 supplemented with 0.3% bovine serum albumin (BSA) and 5 [micro]g/mL cytochalasin B. After enucleation, oocytes were stained with 5 [micro]g/mL bisbenzimide (Hoechst 33342) for 5 min and observed under a Nikon TE-300 inverted microscope equipped with epifluorescence. Oocytes containing DNA materials were excluded from the subsequent experiments. Fibroblasts were trypsinized into single cells and transferred into the perivitelline space of enucleated oocytes. The resulting couplets were equilibrated for 1 min in 0.3 M mannitol solution containing 0.5 mM HEPES, 0.05 mM Ca[Cl.sub.2], and 0.1 mM Mg[Cl.sub.2] in a chamber containing two electrodes. Using a BTX Electro-Cell Manipulator 2001 (Harvard Apparatus, Holliston, MA, USA), couplets were fused with a double DC pulse of 1.5 KV/cm for 45 [micro]s. Following electrical stimulation, reconstructed oocytes were cultured in porcine zygote medium-3 supplemented with 3 mg/mL fatty acid-free BSA and 5 [micro]g/mL cytochalasin B for 3 h in order to suppress extrusion of the second polar body.

On the next day of SCNT, reconstructed embryos were surgically transferred to naturally cycling surrogate sows on the second day of standing estrus. Abdominal ultrasonograpy to test for pregnancy was performed at days 25 to 30 after the embryo transfer. Thereafter, pregnant recipient were examined by ultrasound weekly. At near full-term birth, fetal heartbeat or movement was monitored by Doppler ultrasonography.

Isolation of homozygous [alpha]GT KO cells

Fibroblasts were cultured from ear skin biopsies of the heterozygous [alpha]GT KO piglets as previously reported [2]. Selection of homozygous [alpha]GT KO cells derived from LOH was performed as described elsewhere [11,12]. Briefly, 5 x [10.sup.5] heterozygous [alpha]GT KO cells were washed twice with DPBS, resuspended with 1 mL of DPBS containing 4 [micro]g biotin-conjugated IB4-lectin (EY Laboratories, San Mateo, CA, USA) in a 1.5-mL tube, and incubated for 1 h on ice with tapping the tube to prevent precipitation of the cells. After washing cells twice with DPBS by centrifugation, 4 mg of Dynabeads M-280 Streptavidin (Invitrogen, USA) was added to the pellet and the resulting mixture was again incubated as same as the IB4-lectin treatment. Then, the tube was placed on a magnet for 2 to 3 min, and the supernatant was transferred to a new tube on a magnet. This procedure was repeated three times. The final supernatant was cultured in DMEM containing 10% (v/v) FBS in 60-mm culture dish for 10 to 14 days until colonies reached approximately 2 mm in diameter. The culture dish containing fibroblast colonies was washed twice with DPBS and covered with dimethylpolysiloxane (DMPS). Colonies were dissociated by injecting 30 [micro]L of 0.25% trypsin-EDTA on top of each colony underneath DMPS using micropipette. After incubation for 5 min at 39[degrees]C, fibroblasts dissociated from each colony were transferred onto 0.1% gelatincoated 24-well culture dish for clonal culture. Clones of homozygous [alpha]GT KO were analyzed by PCR as previously reported [2]. Briefly, genomic DNA was prepared from fibroblasts using DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Using Maxime PCR Premix Kit (Intron Biotechnology, Seongnam, Korea) in 20 [micro]L reaction volume, amplification of the target gene was carried out using forward 5'-AGAGGTCGTGACCATAACCAGAT-3' and reverse 5'-AGCCCATGCTGAACATCA[ALPHA]GTC-3' primers. Conditions for PCR were as follows: 30 cycles of 15 s at 94[degrees]C, 30 s at 65[degrees]C, 10 min+20 s increase/cycle at 68[degrees]C; and one final cycle of 7 min at 68[degrees]C. Amplification products (7.4 and 9.2 kb as shown in Figure 1) were analyzed by 0.4% agarose gel electrophoresis. Colonies that LOH was confirmed by PCR were used for SCNT to produce homozygous [alpha]GT KO pigs.

Karyotypic analysis

Karyotype analysis of homozygous [alpha]GT KO fibroblasts was performed by GenDix, Inc (Seoul, Korea), a commercial provider of karyotyping. Briefly, 200 [micro]L of colecemid (Invitrogen, USA) was added to cells and incubated overnight at 39[degrees]C in 5% C[O.sub.2]. Cells were trypsinized, harvested, and centrifuged for 5 min at 400xg. After 10 [micro]L of hypotonic solution (0.075 M KCl) was added to the cell pellet, cells were resuspended, incubated for 20 min at 39[degrees]C in 5% C[O.sub.2], and centrifuged for 5 min at 400xg. Then, hypertonic solution was removed, and 500 [micro]L Carnoy's fixative (methanol:acetic acid = 3:1) was added and mixed with the cells. After fixative containing fibroblasts was dropped and spread on clean glass slides, the slides were baked 60[degrees]C for 30 min and treated with 50% [H.sub.2][O.sub.2] for 3 min. Finally, followed by karyotypic analysis following Giemsa staining (GTG banding) was performed using ChIPS-Karyo (Chromosome Image Processing System, GenDix, Seoul, Korea).

Flow cytometry analysis

To evaluate the expression levels of [alpha]Gal epitope on cell surface, fibroblasts obtained from wild type, heterozygous [alpha]GT KO, and homozygous [alpha]GT KO pigs were analyzed by flow cytometry. Cultured cells were harvested, washed with DPBS and resuspended at 7x[10.sup.4]/mL. Then cells were labeled with fluorescein isothiocyanate (FITC)-conjugated IB4 and analyzed using BD FACSCalibur Cell Analyzer (BD Biosciences, San Jose, CA, USA).

Southern blot

Southern blot hybridization was performed using DIG High Prime DNA Labeling and Detection Kit 2 (Roche Molecular Systems, Pleasanton, CA, USA). Ten micrograms of genomic DNA were digested with a Bst EII (Takara, Shiga, Japan) overnight at 60[degrees]C. As represented in Figure 1, the digestion generates a 6.8 kb fragment in the wild allele and an 8.1 kb fragment in the targeted allele. Samples were separated on a 0.8% agarose gel. Following electrophoresis, restricted genomic DNA was transferred to a nylon membrane (Roche, Switzerland). Then, the membrane was hybridized with a 520 bp DIG-labeled probe in DIG Easy Hyb solution (Roche Molecular Systems, USA) for 16 h at 53[degrees]C. After the hybridization, membrane was washed twice in 0.5x SSC with 0.1% sodium dodecyl sulfate at 68[degrees]C and exposed to a camera for 10 min.

RESULTS

Production of heterozygote [alpha]GT KO pigs

A total of 2x[10.sup.7] fetal fibroblasts were transfected using [alpha]GT gene-targeting vector, and 87 colonies were obtained after antibiotic selection. Among these colonies, two (2.3% efficiency) were identified as heterozygote [alpha]GT KO. One of the two was used for subsequent SCNT experiment. A total of 559 cloned embryos were produced by SCNT and transferred to five surrogates. Two pregnancies went to term, leading to delivery of three female piglets by cesarean section (Figure 2A). Genomic DNA was extracted from tail tip of piglets and analyzed by PCR. All of three piglets were confirmed to be heterozygote [alpha]GT KO (Figure 2B).

Loss of heterozygosity in skin fibroblasts from heterozygous [alpha]GT KO piglets

After IB4-Dynabead selection, 22 colonies were isolated from 5x[10.sup.5] cultured fibroblasts obtained from heterozygous [alpha]GT KO cloned piglets. Based on PCR analysis, 15 colonies were confirmed to have LOH with biallelic disruption of [alpha]GT presumably by mitotic recombination (68.2% LOH efficiency). One of the colonies was chosen for expansion of cells by subculture and subjected to karyotypic analysis. Subcultured homozygous [alpha]GT KO fibroblasts demonstrated normal porcine karyotype (Figure 3) and subsequently used for SCNT.

Production of homozygous [alpha]GT KO pigs

Using the same procedure for the production heterozygous [alpha]GT KO pigs, a total of 729 cloned embryos were produced and transferred to four surrogates. Two pregnancies went to term, leading to delivery of four piglets with three viable and one stillborn by cesarean section (Figure 4A). The stillborn piglets had no gross clinical and anatomical abnormalities, and the cause of death was not clear. As represented in Figure 4B and 4C, genomic DNA analyses by PCR and Southern blot showed that all of the piglets were homozygotes with disruption in both alleles of [alpha]GT gene.

Flow cytometric analysis of [alpha]Gal expression on fibroblasts from homozygous [alpha]GT KO pigs

To examine the expression of [alpha]Gal epitopes on cell membrane, ear skin fibroblasts obtained from wild type, heterozygous, and homozygous [alpha]GT KO piglets were reacted with FITC-conjugated IB4 lectin. As shown in Figure 5, unlike wild type and heterozygous [alpha]GT KO cells, no fluorescence was observed from homozygous [alpha]GT KO fibroblasts, suggesting [alpha]Gal antigen was not expressed on cell surface of homozygous [alpha]GT KO fibroblasts.

DISCUSSION

In the current study, we demonstrated the procedure that heterozygous [alpha]GT KO piglets were produced by conventional genetargeting technique (Figure 2), and subsequently homozygous [alpha]GT KO cells that LOH had occurred were isolated from postnatal heterozygous [alpha]GT KO miniature pig ear-skin fibroblasts. Even after extended in vitro culture for induction of LOH, these cells possessed normal karyotype (Figure 3). Ultimately, homozygous [alpha]GT KO cells gave rise to the production of homozygous piglets using second round cloning (Figure 4). Although the previous study has shown that homozygous [alpha]GT KO could be produced by IB4-Dynabead selection of LOH in heterozygous fetal fibroblasts [11], one drawback of the study is sacrificing heterozygous pigs due to collection of fetuses before the term. Therefore, it needs to be determined whether postnatal fibroblast also could be utilized for the production of homozygous [alpha]GT KO pigs by IB4-Dynabead selection. In this way, homozygous KO pigs could be produced in addition to preservation of heterozygous Animals.

The present study demonstrated that [alpha]GT gene could be spontaneously mutated as a result of LOH in postnatal heterozygous [alpha]GT KO ear skin fibroblasts. During cell division following genetic mutation, LOH could occur as a consequence of mitotic recombination [13]. Later, homozygous [alpha]GT KO pigs were produced from the cells carrying such type of genetic recombination. Flow cytometric analysis using FITC-conjugated IB4 confirmed that the existential level of [alpha]Gal epitope on cell membrane of homozygous [alpha]GT KO piglet was similar to that of unstained cells (Figure 5). The rate of mitotic recombination is dependent on the homology of homologous chromosomes [14,15]. Although we used Minnesota miniature pigs, a nearly inbred miniature pig strain, to isolate homozygous [alpha]GT KO fibroblasts, the recovery rate of LOH in the present study was 0.003% (15 PCR-positive homozygous [alpha]GT KO colonies from 5x105 heterozygous [alpha]GT KO fibroblasts) comparable to the report from Fujimura et al [11]. Even though neonatal fibroblasts were used for the induction of LOH, cloning efficiency of the present study was 0.41% (3 piglets from 729 transferred cloned embryos) which was also comparable to the previous report using fetal fibroblasts [11].

All three live piglets produced by recloning were homozygotes for [alpha]GT KO. We took advantage of applying a new technique in colony picking to increase homogeneous cell population. Due to the nature that a disruption of the [alpha]GT gene is analyzed by PCR, a contamination of heterozygous [alpha]GT KO cells into homozygous [alpha]GT KO colony may cause pseudo-negative results from an undesirable amplification of wild type allele of heterozygous [alpha]GT KO cells. Therefore, it would be important to strictly isolate individual colonies without such contamination. In the present study, we used the oil-cover method using DMPS to decrease pseudo-negative colonies. Before picking colonies by trypsinization, cells were covered with mineral oil, and the viscosity of oil prevented cells that were detached by enzyme treatment from being scattered. Consequently, it was possible to isolate homogenous colonies without contamination.

The present study demonstrated that postnatal skin biopsy and subsequent selection for LOH could be utilized for the production of homozygous [alpha]GT KO piglets. Such procedure could save time and cost for the production of homozygote KO animals by breeding, especially in species that requires substantial length of gestation.

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

https://doi.org/10.5713/ajas.16.0010

ACKNOWLEDGMENTS

This research was supported by a grant (PJ011375) from the Next-Generation BioGreen 21 Program, Rural Development Administration, a grant (2014034046) supported by the Bio & Medical Technology Development Program, and a grant (20090093829) supported by the Priority Research Centers Program through National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning.

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[7.] Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002;20: 251-5.

[8.] Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1, 3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089-92.

[9.] Ramsoondar JJ, Machaty Z, Costa C, et al. Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1, 2-fucosylosyltransferase. Biol Reprod 2003;69:437-45.

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Young June Kim (1,2), Kwang Sung Ahn (1), Minjeong Kim (1), Min Ju Kim (1), Jin Seop Ahn (1), Junghyun Ryu (1), Soon Young Heo (1), Sang-Min Park (1), Jee Hyun Kang (1), You Jung Choi (1), and Hosup Shim (1,3,4) *

* Corresponding Author: Hosup Shim

Tel: +82-41-550 3865, Fax: +82-41-559-7839, E-mail: shim@dku.edu

(1) Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea

(2) Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang 25354, Korea

(3) Institute of Tissue Regeneration Engineering, Dankook University, Cheonan 31116, Korea

(4) Department of Physiology, Dankook University School of Medicine, Cheonan 31116, Korea

Submitted Jan. 5, 2016; Revised Mar 9, 2016; Accepted Mar 29, 2016

Caption: Figure 1. A diagram of gene-targeting vector. Arrows, polymerase chain reaction primers to detect homologous recombination; Bar, probe for Southern blot analysis.

Caption: Figure 2. Production of heterozygous [alpha]GT KO piglets. A, Heterozygote [alpha]GT KO piglets produced by somatic cell nuclear transfer. B, polymerase chain reaction analysis for [alpha]GT gene-targeting. [alpha]GT, alpha-1, 3-galactosyltransferase; KO, knockout.

Caption: Figure 3. Karyotype of homozygous [alpha]GT KO fibroblasts obtained by LOH. Cells contained a normal karyotype of female swine (2n = 38, XX). [alpha]GT, alpha-1,3-galactosyltransferase; KO, knockout; LOH, loss of heterozygosity.

Caption: Figure 4. Production of homozygous [alpha]GT KO piglets. A, Homozygous [alpha]GT KO piglets produced by somatic cell nuclear transfer; B, PCR analysis for [alpha]GT gene-targeting; C. PCR analysis for [alpha]GT gene-targeting. [alpha]GT, alpha-1, 3- galactosyltransferase; KO, knockout; PCR, polymerase chain reaction.

Caption: Figure 5. Flow cytometric analysis of fibroblasts from [alpha]GT-deficient piglets. This histogram plots a single parameter (FITC intensity, horizontal axis) against the number of cells detected (vertical axis). Thick solid line, wild type cells; Thick dotted line, heterozygous [alpha]GT KO cells; Thin dotted line, homozygous [alpha]GT KO cells; Thin solid line, unstained cells. [alpha]GT, alpha-1,3- galactosyltransferase; KO, knockout; FITC, fluorescein isothiocyanate.
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Author:Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Ahn, Jin Seop; Ryu, Junghyun; Heo, Soo
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Mar 1, 2017
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