BIOLOGY OF REPRODUCTION 78, 425–431 (2008) Published online before print 14 November 2007. DOI 10.1095/biolreprod.107.065185
Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1 Xi Jun Yin,3 Hyo Sang Lee,3 Xian Feng Yu,3 Eugene Choi,7 Bon Chul Koo,5 Mo Sun Kwon,5 Young S. Lee,4 Su Jin Cho,4 Guang Zhen Jin,4 Lyoung Hyo Kim,6 Hyoung Doo Shin,6 Teoan Kim,5 Nam Hyung Kim,7 and Il Keun Kong2,4,8 Department of Animal Science and Technology,3 Sunchon National University, Sunchon 540-742, South Korea Division of Applied Life Science,4 Gyeongsang National University, Jinju 660-701, South Korea Department of Physiology,5 Catholic University of Daegu School of Medicine, Daegu 705-718, South Korea Department of Genetic Epidemiology,6 SNP Genetics Inc., Seoul 153-801, South Korea Department of Animal Sciences,7 Chungbuk National University, Cheongju 361-763, South Korea Institute of Agriculture and Life Science,8 Gyeongsang National University, Jinju 660-701, South Korea than rodents or other laboratory mammals [2]. Genetic manipulation of the cat is hampered by a lack of suitable technologies, such as embryonic stem cells (ESs), which are routinely used to generate targeted mutations in the mouse. Cloning through somatic cell nuclear transfer (SCNT) is a potential alternative approach in species for which ES technologies are unavailable. The SCNT technique has been used to produce cloned and transgenic cattle [3], sheep [4, 5], goats [6], and pigs [7, 8]. In order to improve the efficiency of transgenic animal production by SCNT, a method for screening both genetically modified nuclear donor cells and reconstructed embryos before embryo transfer to the surrogate females is critical. Green fluorescent protein (GFP) selection of donor cells has been used to produce transgenic offspring by SCNT in mice [9], pigs [10], and cattle [11]. A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases [1]. As a species, the domestic cat exhibits several distinctive characteristics, one of which is a close genetic relationship to the human genome [2, 12]. The feline gene map displays a higher level of systemic conservation with humans than rodents or other laboratory mammals [2]. The efficient use of nuclear transfer that is associated with the genetic modification of donor cells in domestic cats would substantially improve the efficacy of the production of genetically identical cats that may carry genes for the study of specific human disorders [1]. In addition, these methods could be applied to the production of genetically engineered designer pets with selected favorable attributes, such as an allergen-free cat [1]. The current study was designed to evaluate the feasibility of producing transgenic cats expressing RFP as a reporter gene. In this study we used the SCNT technique to produce transgenic cats that ubiquitously express RFP. We used a Moloney murine leukemia virus-based pseudotyped retroviral vector system in which recombinant viruses are packaged with vesicular stomatitis virus G glycoprotein [13] to deliver the RFP transgene to the cat somatic cells. The significance of this work stems from its precedence: it is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.
ABSTRACT A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean ¼ 16 6 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats. assisted reproductive technology, cat, cloned, cloned animal, developmental biology, red fluorescence protein, somatic cell nuclear transfer, transgenic, transgenic animal
INTRODUCTION Domestic cats have been proposed as a biomedical model for the study of several human diseases [1]. Domestic cats are phylogenetically close to humans, and the feline gene map displays a higher level of systemic conservation with humans 1
Supported by a grant from the Korea Science and Engineering Foundation (KOSEF; grant M10525010001-05N2501-00110), funded by the Korean government (MOST). E.C., Y.S.L., S.J.C., and G.Z.J. were supported by scholarships from the Post BK21 Program. 2 Correspondence: Il Keun Kong, Division of Applied Life Science, Gyeongsang National University, Jinju 660-701, GyeongNam Province, South Korea. FAX: 82 55 756 7171; e-mail:
[email protected]
MATERIALS AND METHODS
Received: 23 August 2007. First decision: 17 September 2007. Accepted: 2 November 2007. Ó 2008 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
Source of Reagents All of the chemicals were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO) unless otherwise stated.
425
426
YIN ET AL.
FIG. 1. Schematic diagram of the LHCRW provirus. Approximate positions of the Southern blotting probe and the PCR primer set for the detection of the DsRed2 (or RFP) gene sequence are indicated above the vector. The drawing is not to scale. LTR, long terminal repeat; HygR, hygromycin-resistant gene; CMVp, cytomegalovirus promoter; DsRed2, red fluorescent protein gene; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.
Animal Care and Use The Stroller female cats that were used as oocyte donors were housed in stainless steel cages measuring 0.9 3 0.7 3 0.65 m and were provided with dry food and water ad libitum. They were maintained in an environmentally controlled room on a 14L:10D photoperiod cycle with light onset at 0600 h. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Sunchon National University.
with 5% CO2 in air in DMEM containing 10% FBS until 90% confluence was observed. Cells then were trypsinized and reconstituted at 1 3 106 cells/ml, grown to confluence, and frozen as zero passage cells (1 vial/35-mm dish) in DMEM (Gibco BRL) containing 10% dimethylsulfoxide and 10% FBS. Cells
Ovary Recovery and Oocyte Maturation The ovarian follicular development of mature cats was stimulated by injection with 200 IU eCG (Daesung, Seoul, South Korea), and oocyte maturation was induced 4 days later by treatment with 100 IU hCG (Daesung). Ovaries were removed 24 h after hCG injection by routine ovariohysterectomy and were minced in m-Tyrode medium-Hepes using a scalpel blade to release cumulus oocyte complexes [14]. Collected oocytes were matured in TCM199 (M-7528) supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen, Carlsbad, CA) and 1% penicillin G/streptomycin (P-4333) for 4 h at 388C in an atmosphere of 5% CO2 in air.
Construction of the Retrovirus Vector and Virus Production A plasmid (pLHCRW) containing a retrovirus vector sequence was constructed by replacing the TRE sequence of pRevTRE (Clontech, Mountain View, CA) with a fragment containing the CMV promoter, DsRed2 gene, and WPRE (woodchuck hepatitis virus posttrancriptional regulatory element) sequences (Fig. 1). The CMV promoter and the DsRed2 gene were derived from pLNCX and pDsRed2-C1, respectively (Clontech). The WPRE sequence from woodchuck hepatitis virus 2 genomic DNA (GenBank accession number M11082) was introduced following the strategy of Zufferey et al. [15]. Retrovirus-producing cells were constructed following the procedure described by Koo et al. [16]. PT67 cells (Clontech) were transiently transfected with pLHCRW; the resultant LHCRW viruses were harvested and applied to the GP2–293 cells (Clontech). PT67 are retrovirus packaging cells that are characterized by the expression of the Gibbon ape leukemia virus envelope gene, whereas GP2–293 cells are specialized to express the gag and pol genes of Moloney murine leukemia virus. LHCRW-infected GP2–293 cells were selected with hygromycin (150 ll/ml) for 2 wk, and the resultant HygR (hygromycin-resistant) cells were transfected with pVSV-G (Clontech) to express VSV-G protein. Viruses were harvested 48 h after transfection. All virus-producing cells were grown at 378C in a 5% CO2 incubator in Dulbecco modified Eagle medium (DMEM) with 4.5 g/l glucose (Gibco BRL, Grand Island, NY) supplemented with fetal calf serum (10%), penicillin (100 lg/ml), and streptomycin (100 lg/ml). The virus-containing medium that was harvested from the virus-producing cells was filtered through a 0.45-lm pore size filter and used to infect cat fibroblast cells. LHCRW-infected fibroblasts were selected with hygromycin (150 ll/ml) for 2 wk.
Establishment of Cat Fibroblast Cell Lines Several fibroblast cell sublines were established from single, 6-mm skin biopsies taken aseptically from a male, odd-eyed, all-white cat. Primary cultures were obtained by mincing dermal tissue with sterile scissors in a 35mm Petri dish (Nunc, Roskilde, Denmark) and culturing the fragments at 388C
FIG. 2. Expression of RFP in cat fibroblasts and during SCNT. Cells were grown to semiconfluence (a, b) and then trypsinized (c, d) to select single cells emitting a bright red color prior to SCNT. Panels a, c, b, and d were photographed under brightfield light (a, c) and fluorescent light (b, d). Injection of an RFP-positive cell into an enucleated oocyte under conventional light (e) and fluorescence light (f) is shown. Original magnification 3200.
427
PRODUCTION OF CLONED TRANSGENIC RED CATS TABLE 1. In vitro and in vivo development of embryos cloned from RFP transgenic fibroblasts in cats. Oocyte source
No. of oocytes
No. fused (%)
No. cultured
No. of cleaved (%)
No. of blastocyst (%)
No. of positive blastocyst (%)
No. of cells in blastocyst/ no. of counted blastocyst*
In vitro In vivo
456
261 (57.2)
258 46/71**
202 (78.3)a 32 (69.6)a
8 (3.1)a 3 (6.5)b
8 (100) 3 (100)
78.2 6 45.5/8a 508 6 477.9/3b
a,b
Values in the same column with different superscripts differ significantly (P , 0.05). Number of cells in blastocysts at Day 7 of culture (mean 6 SEM). ** Number of recovered cloned embryos per total transferred couplets seven days after transfer. *
were thawed and cultured for RFP gene transfection. RFP-transfected cells yielding confluent growth after culture in serum-free medium for 3 days were used for SCNT [14]. Single RFP-transfected cells were selected under an inverted microscope equipped with a G-2A filter (EX: 510–560 nm, BA: 590 nm; Fig. 2).
Somatic Cell Nuclear Transfer The SCNT protocol was described previously [14]. Briefly, cumulus cells were removed from oocytes by gentle pipetting in TCM199 supplemented with 0.1% hyaluronidase. The denuded oocytes were cultured in TCM199 supplemented with 0.2 lg/ml demecolcine for 1 h and then placed in TCM199 containing 5 lg/ml cytochalasin B and 0.2 lg/ml demecolcine. The protruding first polar body and chromatin plate were removed with a beveled micropipette mounted on micromanipulators (Narishige, Tokyo, Japan) while being viewed with an inverted microscope (Nikon, Tokyo, Japan) using Hoffman modulation contrast optics. Successful enucleation was confirmed by staining the oocytes with Hoechst 33342 (5 lg/ml) and observing them with a fluorescent microscope under ultraviolet light. Fibroblast donor cells then were dissociated using 1% trypsin-EDTA and placed in Ca2 þ-free and Mg2 þ-free DPBS supplemented with 0.3% BSA (fatty acid free). Micromanipulation was used to place a midsized (20–25 lm) single donor cell nucleus into the perivitelline space of each enucleated oocyte. Cytoplast-cell couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Mg2 þ and were transferred to an electrofusion chamber containing the same medium. Cell fusion was induced by applying 2.0 kV/cm in 20-lsec DC pulses (23) delivered by an electro cell fusion generator (Nepagene, Chiba, Japan). Couplets were removed from the fusion chamber, washed, and incubated in TCM199 supplemented with 0.3% BSA at 388C in an atmosphere of 5% CO2 in air. At 1 h after electrofusion, fused couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Ca2 þ and 0.1 mM Mg2 þ, placed into a fusion chamber containing the same medium, and electropulsed by applying 1.0 kV/cm in 20-lsec DC pulses, 0.1 sec apart (23). Activated couplets then were washed and incubated for 4 h in TCM199 supplemented with 0.3% BSA and 2 mM 6-DMAP at 388C in an atmosphere of 5% CO2 in air.
In Vitro and In Vivo Development of Cloned Embryos Derived from RFP Transgenic Fibroblasts To culture the in vitro embryos, activated cytoplast-cell couplets were grown in 50-ll droplets of m-Tyrode medium [17] supplemented with 1% MEM nonessential amino acid and 3 mg/ml BSA (Tyrode 1) for 3 days before being changed to m-Tyrode medium with 1% nonessential amino acid, 2% MEM BME amino acids (EAA), and 10% FBS (Tyrode 2) for the remaining 4 days of culture. The frequency of cleavage was assessed on Day 3, and the incidence of blastocyst development was visually determined on Day 7. To culture and collect the in vivo embryos, three female cats were treated with eCG and hCG as described below. At 36 h after treatment with hCG, activated
cytoplast-cell couplets were surgically transferred into the oviduct of the recipient. Seven days after transfer, blastocysts were flushed from the excised uterus using TALP-Hepes buffered saline containing 0.3% BSA (A-9647).
Evaluation of Cell Number Blastocysts that were derived from the in vitro and in vivo development were fixed for 15 min at room temperature in PBS containing 3.7% paraformaldehyde and 0.1% BSA before being stained with Hoechst 33342 (20 lg/ml). Embryos then were placed in a drop of glycerol on a slide, covered with a cover slip, and examined for nuclei staining using a fluorescence microscope.
Cloned Embryo Transfer into Synchronized Recipients Activated cytoplast-cell couplets were cultured in 50-ll droplets of TCM199 with 4 mg/ml BSA under mineral oil in a humidified atmosphere of 5% CO2 in air for 24 h. The number of embryos at the two- to four-cell stage were counted and transferred into the oviducts of healthy, mature, female domestic cats that had been synchronized by an injection of 100 IU eCG followed by an injection of 100 IU hCG 96 h after ovulation. The cloned embryos were transferred approximately 30 h after hCG injection. Each recipient was anesthetized with acepromazine maleate (0.025 mg/kg; Sedaject; Samwoo) and ketamine (5 mg/kg; Daesung) before laparotomy. Only cats that had fresh ovulation sites in their ovaries were used as recipients. Pregnancies were detected on Day 40 or 45 after transfer using a SONOACE 900 ultrasound scanner (Medison Co. Ltd., Seoul, South Korea) with an attached 7.0-MHz linear probe.
Microsatellite Analysis of Cloned Cats TG cats obtained by SCNT were subjected to parentage analysis. DNA was extracted from tissue collected from newborn cats, surrogate recipients, and donor cells. Nine feline DNA microsatellite markers (FCA229, FCA290, FCA305, FCA441, FCA078, FCA201, FCA224, FCA170, and FCA304) were used to confirm the genetic identity of the cloned cats and the donor fibroblast cells [14]. Genomic DNA was amplified by PCR using nine feline microsatellite primers that were fluorescently tagged with HEX (Applied Biosystems, Foster City, CA). All of the PCR products were run on a 3100 Genetic Analyzer, and the allele sizes were calculated using Genescan software (Applied Biosystems).
Genomic DNA Analyses Genomic DNA was extracted from cats using the G-DEX Genomic DNA Extraction Kit (iNtRON Biotechnology, Seoul, South Korea). To amplify the RFP gene by PCR, the nucleotide sequence of the pDsRed2-C1 cloning vector (catalogue number 632407; Clontech) was used to design an upstream (5 0 -
TABLE 2. In vivo development of embryos cloned from RFP transgenic fibroblasts in cats. Surrogate mother (S)
No. of SCNT embryos transferred (mean)
S1 S2 S3 S4–S11 Total * ND, Not detected.
13 14 9 140 176 (16 6 7.5)
Pregnancy
Delivery (at Days)
Yes Yes
Abortion (ND*) 2 Kittens (65; 1 Liveborn and 1 stillborn, denoted TG-A and TG-B) 1 Kitten (66; 1 Liveborn, denoted TG-C)
Live; RFP expression
2/11 (18.2%)
3/3 (100%)
Yes No 3/11 (27.3%)
Current status and RFP expression 1 Live; RFP expression in both cats
428
YIN ET AL.
FIG. 3. RFP expression in two transgenic cloned cats (left) and the control cat (right) 60 days after birth. a) Visible light image. a 0 ) Fluorescence image. The red fluorescence in a 0 was produced by illumination with 540 6 20 nm and detected using an emission filter with a maximal transmittance wavelength of 600 6 25 nm. GTTCCAGTACGGCTCCAAGGTGTA-3 0 ) and downstream (5 0 -ATGGTGTAGTCCTCGTTGTGGGAG-3 0 ) primer pair corresponding to the pDsRed2C1 nucleotide sequence at positions 804–827 and 1218–1241 (Fig. 1), respectively, yielding a predicted amplification product of 438 base pairs. Each reaction mixture contained 100 ng genomic DNA extract, 10 pmol of each primer, and 10 ll of 23 GoTaq Green Master Mix (Promega, Madison, WI) in a final reaction volume of 20 ll. The initial denaturation was performed at 948C for 5 min, followed by 35 cycles of PCR amplification. The amplification profile consisted of three steps: 948C for 30 sec (denaturation), 588C for 30 sec (annealing), and 728C for 30 sec (extension). After 35 amplification cycles, the samples were maintained at 728C for 7 min to ensure that complete strand extension had taken place. For Southern blot analysis, genomic DNA (25 lg) was digested with KpnI, and fragments containing the full sequence of the RFP gene were separated on a 0.8% agarose gel. For blot analysis, primers for the synthesis of the RFP gene probe were 5 0 -CGCCACCATGGCCTCCTC-3 0 and 5 0 -CAGGAACAGGTGGTGGCG-3 0 , corresponding to pDsRed2-C1 nucleotide sequence positions 606–623 and 1270–1287, respectively (Fig. 1). The probe was synthesized using a PCR DIG Probe Synthesis kit (Roche, Mannheim, Germany), purified by agarose gel electrophoresis, and labeled with digoxigenin before hybridization. Detection of labeled DNA on the positively charged nylon membrane was performed using a DIG luminescent detection kit (Roche).
Detection of Red Fluorescence Protein Red fluorescence of liveborn and stillborn RFP TG cats was detected using excitation-emission filter sets. Light was supplied from a 70-W surgery light equipped with an excitation filter (HQ 540/40; Chroma Technology Corp., Rockingham, VT). Red fluorescence emission from the cat and organs was detected using a Samsung digital camera (GX10) equipped with emission filters (HQ 600/50; Chroma Technology) that were attached proximal to the camera lens. The filter was easily removed for imaging with white light illumination.
Statistical Analysis Each experiment was repeated at least three times. The effects of different culture media on activated oocyte cleavage and development to the blastocyst stage were analyzed by the chi-square test. The blastocyst cell number and the difference between groups were evaluated by the Student t-test. The level of significance was set at P , 0.05. TABLE 3. Body and placental weights at birth of cloned TG cats. Cloned cat TG-A TG-B TG-C a
ND, Not determined.
Body weight (g)
Placenta weight (g)a
110 122 136
20 ND 29
FIG. 4. RFP expression in the body and organs of the TG-B stillborn cloned cat (right) and non-TG cat (left). Photos were taken under visible light (left frames) and fluorescence light (right frames): hair (a, a 0 ), muscle (b, b 0 ), brain (c, c 0 ), heart (d, d 0 ), kidney (e, e 0 ), stomach and intestine (f, f 0 ), bronchus and lung (g, g 0 ), liver (h, h 0 ), spleen (i, i 0 ), tongue (j, j 0 ), and excrement (k, k 0 ). The ruler is marked in centimeters.
429
PRODUCTION OF CLONED TRANSGENIC RED CATS TABLE 4. Microsatellite analysis of donor, cloned TG, and surrogate mother (S) cats using nine feline specific microsatellite markers. Feline microsatellite markers Cloned cat
FCA078
FCA170
FCA201
FCA224
FCA229
FCA290
FCA304
FCA305
FCA441
Original donor cells TG-A TG-B TG-C S2 S3
200/200 200/200 200/200 200/200 202/202 184/200
109/113 109/113 109/113 109/113 89/109 118/122
140/150 140/150 140/150 140/150 140/156 140/154
150/150 150/150 150/150 150/150 158/158 150/170
171/171 171/171 171/171 171/171 151/169 167/169
212/218 212/218 212/218 212/218 214/214 213/213
110/110 110/110 110/110 110/110 110/112 110/116
185/191 185/191 185/191 185/191 195/195 194/196
155/159 155/159 155/159 155/159 159/159 155/159
RESULTS In Vivo and In Vitro Developmental Potential of Cloned RFP TG Embryos We monitored in vitro development and RFP expression in reconstructed embryos. Within 1 h after fusion, strong fluorescence was detected in reconstructed oocytes. This fluorescence was probably not derived from transcription of the donor cell genome; rather, it probably originated from the cytoplasm of the donor cell, which contained RFP mRNA as well as RFP protein. Since the fusion method was employed in this experiment, cytoplasmic material from donor cells was fused into the cytoplasm of oocytes. Fluorescence in NT embryos became weaker at the four-cell and morula stages, similar to results reported for swine [18]. At 2 h after fusion, strong fluorescence was sustained in reconstructed oocytes; however, at 15–48 h after fusion, the fluorescence in NT embryos became weaker or disappeared. After Day 3, cleaved embryos at the two- to four-cell stages started to express RFP again. We compared the in vitro and in vivo developmental competence of embryos generated from RFP-transfected cell lines by SCNT. The incidence of cleavage was similar between in vitro- and in vivo-developed embryos (78.3% vs. 69.6%; Table 1); however, development to the blastocyst stage was significantly higher in in vivo development than in vitro cultures (6.7% vs. 3.0%; P , 0.05). The average cell number
of in vivo blastocysts was significantly higher than that in in vitro blastocysts (508 6 477.9 vs. 78.2 6 45.5; P , 0.05). In our in vitro culture system, the percentage of embryos that developed to the blastocyst stage was only 3%. Eight blastocysts were examined in order to determine the average blastocyst cell number. These data suggest that the in vitro culture media was not appropriate for cat embryo culture. To overcome this problem, we limited the pretransfer culture time to less than 24 h. Production of RFP TG Kittens and Confirmation of the Genetic Identification of Cloned Cats In order to produce RFP TG kittens, cytoplasmic/cell couples were cultured in TCM199 for 24 h and were then assessed for positive developmental cleavage. Cleaved embryos were transferred into the oviducts of recipient cats that were synchronized with the embryos’ developmental stage. A total of 176 cleaved, reconstructed cat embryos were transferred into 11 surrogate mothers (mean ¼ 16 6 7.5 per recipient; Table 2). Three surrogate mothers were confirmed pregnant (27.3%) by ultrasound scans at 40–45 days; S-2 delivered one liveborn and one stillborn kitten (TG-A and TG-B) at 65 days of gestation, and S-3 delivered one liveborn kitten (TG-C) at 66 days of gestation. The body weights of TG-A, TG-B, and TG-C kittens at birth were 110, 122, and 136 g, respectively. The placental weights of the liveborn TG-A and TG-C kittens were 20 and
FIG. 5. PCR and Southern blot analyses of a TG cat. Genomic DNA isolated from various organs was subjected to PCR amplification (I) or Southern blot analysis (II). DNA sample sources used in the PCR analysis were pLHCRW plasmid (P); cultured cat fibroblasts transduced (cellþ) or nontransduced (cell) with LHCRW retrovirus; muscle of non-TG cat (NTM); TG stillborn kitten muscle (M), liver (L), kidney (K), brain (B), large intestine (I), heart (H), skin (S), and testis (T); and TG liveborn kitten placenta (PL). The expected size of the amplified RFP gene product is indicated. In Southern blot analysis, genomic DNA fragments that had been digested with KpnI were electrophoresed and were hybridized with the probe for the RFP gene depicted in Figure 1. Plasmid DNA (pLHCRW) was used as the positive (lane P) control. The expected size of the RFP gene fragment is indicated. M, molecular size marker; N, non-TG cat; T, stillborn TG cat.
430
YIN ET AL.
All of the fibroblasts that emitted a red color under fluorescent light were used as donor cells to make cloned embryos (Fig. 2). At each stage, all of the cloned embryos expressed the RFP transgene; RFP transgene expression in in vitro- and in vivo-derived SCNT embryos was confirmed under conventional and fluorescence light sources. Although the number of cloned embryos was not enough for statistical analysis, each one that was examined positively expressed RFP (100% in each group; Table 1). At 60 days after birth, the transgenic cloned cats and control cat were examined under a portable surgery light fitted with excitation and emission filter sets to assess the expression level of RFP. Red fluorescence was observed in TG-A and TG-C cats, but not in the control cat (Fig. 3). The expression pattern of the RFP transgene was determined in several tissues taken from the TG stillborn kitten (TG-B) and non-TG cat. Red fluorescence was detected in tissues, including the brain, heart, liver, kidney, testis, lung, muscle, intestine, thymus, spleen, seminal vesicle, skin, hair, fat, and cultured blood cells that were isolated from the transgenic cat’s umbilical cord blood; however, no red fluorescence was observed in tissues from control cats (Fig. 4). Under brightfield illumination, the transgenic cats appeared red and were readily distinguished from the wild-type cats; this observation is similar to that described in a previous report [19]. The level of red fluorescence was pronounced and easily apparent in most of the tissues examined, but was relatively subtle in heart tissue. These data are comparable with the RFP expression observed in mice [19], which exhibited lower levels of RFP expression in adipose, lung, spleen, and testis tissue, and no RFP expression in red blood cells. The presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses (Fig. 5). RFP gene expression was detected in muscle and skin from the stillborn kitten (TG-B), but not from the placenta of a non-TG cat.
improved mutant variants may offer greater usefulness in certain applications. Red fluorescent protein represents a type of fluorescent protein that was initially isolated as DsRed from the sea anemone relative, Discosoma [26]. A desired gene can be transferred into the somatic cell by infection with a viral vector or by electroporation with lipid (liposome) or nonlipid (polymer) reagents carrying DNA. For instance, Park et al. [27] used a retroviral vector to produce GFP-cloned piglets, Lai et al. [28] used electroporation to produce 1,3-galactosyltransferase knockout cloned piglets, and Hyun et al. [29] introduced a GFP gene by liposome-mediated gene transfer to produce transgenic cloned piglets. The rate of infection of the RFP gene using the retrovirus vector was more than 80% (Kong et al., unpublished data), which represents a high level of efficacy for cat fibroblast RFP gene infection. In agreement with our result, several previous reports [30–32] showed that not all of the cells that survived after antibiotic selection were transgenic, suggesting a requirement for prescreening SCNT embryos derived from these donor cells prior to embryo transfer into the recipient females. Transgenic cats were examined under a portable, long-wave surgical lamp to assess the expression level of RFP, and all three kittens exhibited red fluorescence. The expression pattern of the RFP transgene in tissues and cells that were taken from the stillborn transgenic cat was determined. Despite high levels of RFP expression, including expression in the brain and muscle, RFP transgenic cats were apparently normal and showed gross behavior similar to control cats. These observations are consistent with those for pCXmRFP1 transgenic mice, which were apparently normal and exhibited fertility, gross behavior, and a lifespan similar to that of wildtype FVB/NJ mice. Analysis of body weight and the length and size of organs, including heart, liver, kidney, and spleen, revealed no significant difference between age- and sexmatched pCX-mRFP1 transgenic mice and wild-type mice [19]. In conclusion, we report for the first time the production of cloned transgenic cats with systemic expression of red fluorescent protein by somatic cell nuclear transfer. Application of the SCNT procedure to produce genetically modified cats would be valuable for generating biomedical models of human disease, as well as for the production of designer pets.
DISCUSSION
REFERENCES
29 g, respectively (Table 3). The placenta of TG-B was not recovered due to a natural delivery at midnight. Analysis of eight feline specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat (Table 4). Analysis of RFP Expression in RFP TG Cloned Embryos and Kittens
The GFP and RFP genes are useful markers for the identification of transgenic nuclear transfer embryos prior to embryo transfer [20]. Use of these reporter genes might significantly enhance the efficiency of transgenic animal production, because expression of these genes allows positive embryos to be selected before they are transferred into the surrogate mother [21]. In addition, the descendants of a transgenic pig expressing GFP will probably provide a variety of genetically marked tissues, which would be very useful for basic research where such marked cells are required [18]. In this study, we achieved the ubiquitous, nontoxic expression of RFP in transgenic cats using the SCNT technique. This method could be offered as an alternative to genetic manipulations, such as lineage tracing in chimeric cats or establishing RFP expression in embryonic stem cell lines. The enhanced green (EGFP) [22], yellow (EYFP) [23], and cyan (ECFP) [24] fluorescent proteins have served as invaluable tools for these purposes for many years; however, the emission spectra of these fluorochromes are relatively close, which makes their visual separation difficult with readily available imaging systems. For these reasons, RFPs such as DsRed [25] and its
1. Gomez MC, Pope CE, Dresser BL. Nuclear transfer in cats and its application. Theriogenology 2006; 66:72–81. 2. Menotti-Raymond M, David VA, Lyons LA, Schaffer AA, Tomlin JF, Hutton MK, O’Brien SJ. A genetic linkage map of microsatellites in the domestic cat (Felis catus). Genomics 1999; 57:9–23. 3. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM. Cloned transgenic calves produced from nonquiescent fetal fibroblasts [see comment]. Science 1998; 280:1256–1258. 4. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King T, Ritchie M, Ritchie WA, Rollo M, de Sousa P, Travers A, et al. Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep [see comment]. Nat Biotechnol 2001; 19:559–562. 5. McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE, Kind AJ. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells [see comment] [erratum appears in Nature 2000; 408:120]. Nature 2000; 405:1066–1069. 6. Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris A, Karatzas CN. Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol Reprod 2001; 64:849–856. 7. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs [see comment]. Nat Biotechnol 2002; 20:251–255.
PRODUCTION OF CLONED TRANSGENIC RED CATS 8. Lai L, Prather RS. Progress in producing knockout models for xenotransplantation by nuclear transfer. Ann Med 2002; 34:501–506. 9. Sato K, Hosaka K, Ohkawa M, Tokieda Y, Ishiwata I. Cloned transgenic mouse fetuses from embryonic stem cells. Human Cell 2001; 14:301–304. 10. Park KW, Lai L, Cheong HT, Cabot R, Sun QY, Wu G, Rucker EB, Durtschi D, Bonk A, Samuel M, Rieke A, Day BN, et al. Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. Biol Reprod 2002; 66: 1001–1005. 11. Bordignon V, Keyston R, Lazaris A, Bilodeau AS, Pontes JH, Arnold D, Fecteau G, Keefer C, Smith LC. Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitrotransfected somatic cells. Biol Reprod 2003; 68:2013–2023. 12. Murphy WJ, Sun S, Chen ZQ, Pecon-Slattery J, O’Brien SJ. Extensive conservation of sex chromosome organization between cat and human revealed by parallel radiation hybrid mapping. Genome Res 1999; 9:1223– 1230. 13. Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells [see comment]. Proc Natl Acad Sci U S A 1993; 90: 8033–8037. 14. Yin XJ, Lee HS, Kim LH, Shin HD, Kim NH, Kong IK. Effect of serum starvation on the efficiency of nuclear transfer using odd-eyed white cat fibroblasts. Theriogenology 2007; 67:816–823. 15. Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73:2886–2892. 16. Koo BC, Kwon MS, Choi BR, Kim JH, Cho SK, Sohn SH, Cho EJ, Lee HT, Chang W, Jeon I, Park JK, Park JB, et al. Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector. FASEB J 2006; 20:2251–2260. 17. Gomez MC, Jenkins JA, Giraldo A, Harris RF, King A, Dresser BL, Pope CE. Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes. Biol Reprod 2003; 69:1032–1041. 18. Lai L, Park KW, Cheong HT, Kuhholzer B, Samuel M, Bonk A, Im GS, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS. Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol Reprod Dev 2002; 62:300–306. 19. Zhu H, Wang G, Li G, Han M, Xu T, Zhuang Y, Wu X. Ubiquitous expression of mRFP1 in transgenic mice. Genesis 2005; 42:86–90. 20. Zhang YH, Pan DK, Sun XZ, Sun GJ, Liu XH, Wang XB, Tian XH, Li Y, Dai YP, Li N. In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells. Zygote 2006; 14:239–247.
431
21. Uhm SJ, Kim NH, Kim T, Chung HM, Chung KH, Lee HT, Chung KS. Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector. Mol Reprod Dev 2000; 57:331–337. 22. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997; 407:313– 319. 23. Hadjantonakis AK, Nagy A. FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter. Genesis 2000; 27:95–98. 24. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 2001; 1:4. 25. Vintersten K, Monetti C, Gertsenstein M, Zhang P, Laszlo L, Biechele S, Nagy A. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. [erratum appears in Genesis 2005; 42:218]. Genesis 2004; 40:241–246. 26. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA. Fluorescent proteins from nonbioluminescent Anthozoa species [see comment] [erratum appears in Nat Biotechnol 1999; 17:1227]. Nat Biotechnol 1999; 17:969–973. 27. Park KW, Cheong HT, Lai L, Im GS, Kuhholzer B, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS. Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein. Anim Biotechnol 2001; 12:173–181. 28. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, et al. Production of alpha 1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295:1089–1092. 29. Hyun S, Lee G, Kim D, Kim H, Lee S, Nam D, Jeong Y, Kim S, Yeom S, Kang S, Han J, Lee B, et al. Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. Biol Reprod 2003; 69:1060–1068. 30. Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, et al. Production of cloned pigs from in vitro systems [see comment]. Nat Biotechnol 2000; 18:1055–1059. 31. Bondioli K, Ramsoondar J, Williams B, Costa C, Fodor W. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol Reprod Dev 2001; 60:189–195. 32. Gong G, Dai Y, Fan B, Zhu H, Zhu S, Wang H, Wang L, Tang B, Li R, Wan R, Liu Y, Huang Y, et al. Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer. Mol Reprod Dev 2004; 69:278–288.
