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Ectopic expression of FGF5s induces wool growth in Chinese merino sheep
Gene 627 (2017) 477–483
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Research paper
Ectopic expression of FGF5s induces wool growth in Chinese merino sheep a,b,c
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Wen-Rong Li , San-Gang He , Chen-Xi Liu , Xue-Mei Zhang , Li-Qin Wang , Jia-Peng Linb,c, Lei Chenb,c, Bin Hanb,c, Jun-Cheng Huangb,c, Ming-Jun Liub,c,⁎ a
College of Life Science and Technology, Xinjiang University, No. 14 Shengli road, Urmuqi, Xinjiang, China Key Laboratory of Genetics, Breeding & Reproduction of Grass-Feeding Livestock, Ministry of Agriculture, No. 468 Alishan road, Urmuqi, Xinjiang, China Key Laboratory of Animal Biotechnology of Xinjiang Institute of Animal Biotechnology, Xinjiang Academy of Animal Science, No. 468 Alishan road, Urmuqi, Xinjiang, China
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A B S T R A C T Fibroblast growth factor 5 (FGF5) has been recognized as an inhibitor to cease animal hair growth, while in contrary, FGF5 short alternative transcript (FGF5s) can induce hair growth by antagonizing FGF5 function. To investigate the role of FGF5s in wool growth in Chinese Merino sheep, we generated transgenic sheep of ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. Totally 20 transgenic sheep were obtained and 12 were alive after birth. Characterization of the transgene revealed that the transgenic sheep showed variety of integrant, ranged from 2 to 11 copies of transgene. The ectopic expression of FGF5s was observed in all transgenic sheep. Further study on the effect of ectopic expression of FGF5s revealed that the wool length of transgenic sheep were significantly longer than that of non-transgenic control, with 9.17 cm of transgenic lambs versus 7.58 cm of control animals. Notably, besides the increase of wool length, the yearling greasy fleece weight was also concordantly greater than that of wild-type (p < 0.01), with 3.22 kg of transgenic sheep versus 2.17 kg of control lambs (p < 0.01) in average. Our results suggested that overexpression of FGF5s could stimulate wool growth and resulted in increase of wool length and greasy wool weight.
1. Introduction Fine wool sheep had played critical role in textile and cloth industry in mankind history. Wool productivity and quality are essential factors for improvement of the profit of fine wool production. As a critical economic trait, wool fiber length is closely associated to wool productivity and quality. Under the similar wool density and fineness, the wool length has positive correlation to wool yield. Additionally, wool length is the secondly important quality trait coming after the fineness in wool textile industry, which influences the textile product quality and determines the manufacture system. Therefore, a considerable effort has been strengthened to increase the wool length by traditional genetic approaches in fine wool sheep breeding. In mice, a recessive phenotype known as angora(go), is featured by approximately 50% longer hair than wild type. The insight into the mechanism of “go” phenotype revealed that the go mice had longer anagen VI phase. It is the abnormal long anagen VI phase that results in the long hair phenotype (Konyukhov and Berdaliev, 1990). The subsequent research identified a null mutation of FGF5 gene which
contributed to the angora phenotype, and indicated that the transition between anagen and catagen is normally regulated by FGF5 (Hébert et al., 1994). Thereafter, by utility of state-of-art genomic approaches, substantial causal mutations in various species have been found to be related to hair length. Interestingly, all testified or candidate genes responsible for hair length were attributed to FGF5. So far, a number of spontaneous loss-of-function mutations of FGF5 associated to hair length have been identified in human (Higgins et al., 2014), mice (Nesterova et al., 2010; Mizuno et al., 2011), dogs ((Housley and Venta, 2006; Dierks et al., 2013), cats (Drögemüller et al., 2007)) and donkeys (Legrand et al., 2014). It is evidenced that FGF5 is the leading candidate gene for hair length variation. In sheep, FGF5 gene has been under intense selection (Kijas et al., 2012). However, no causal mutations have yet been identified to associate with wool length in fine wool sheep. FGF5 is a member of FGF family with 23 related genes and the role of FGF5 in hair growth is recognized as inhibitor to anagen phase. Sheep FGF5 was consisted by 3 exons and 2 introns spanned 21,743 bp genomic sequence (Lihua et al., 2015). By alternative splicing, FGF5
Abbreviations: FGF5, The fibroblast growth factor 5 gene; FGF5s, Fibroblast growth factor 5-short; FGF, The fibroblast growth factor; FGFR1, The fibroblast growth factor receptor1; FGFR2, The fibroblast growth factor receptor2 ⁎ Corresponding author at: Key Laboratory of Genetics, Breeding & Reproduction of Grass-Feeding Livestock, Ministry of Agriculture, No. 468 Alishan road, Urmuqi, Xinjiang, China. E-mail address: [email protected] (M.-J. Liu). http://dx.doi.org/10.1016/j.gene.2017.06.037 Received 5 May 2017; Received in revised form 15 June 2017; Accepted 20 June 2017 Available online 28 June 2017 0378-1119/ © 2017 Published by Elsevier B.V.
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2.3. Preparation of lentiviral particles and generation of transgenic sheep
gene produces two transcripts, the full length FGF5 with complete three exons and short alternative transcript (FGF5s) without exon2 and part of exon3. The full length transcript which functions to inhibit the activation of derma papilla cells proliferation and synthesis of hair fiber during anagen, and promotes the transition from anagen to catagen (Ota et al., 2002). The FGF5s has no inhibitory function in hair growth, whereas it binds to the FGF receptor1 (FGFR1) and 2 (FGFR2) to compete the FGF5 binding and serves as an antagonist to inhibit FGF5 function (Ozawa et al., 1998). Previous studies addressed that FGF5s could relieve hair growth inhibition in mice and cashmere goats(Suzuki et al., 2000; He et al., 2015) In contrast to other animals, the wool growth in fine wool sheep is distinct by life-long growing without seasonal spontaneous hair removal. So the function of FGF5s in fine wool sheep is still far from understood. Given that the high similarity between Merino sheep FGF5 gene sequence and other mammalian orthologs was observed in our previous study (Lihua et al., 2015), we postulated that the wide type FGF5 could be likely to function inhibitory effect in fine wool sheep, and FGF5s could serve as its antagonist to stimulate wool growth. Nowadays, with the fast development of genome modification approaches, scientists have applied the versatile gene manipulating approaches to modify genes associated with specific traits. Knockout or disruption of FGF5 by gene targeting in mice or cashmere goats resulted in increase of hair or cashmere length (Hébert et al., 1994; Wang et al., 2016). Lentiviral transgene delivery system offers high efficient means in producing transgenic animals (Hofmann et al., 2003; Pfeifer, 2004; Amendola et al., 2005). Our previous study documented that lentiviral transgenesis achieved high efficiency and widespread expression of EGFP in transgenic sheep (Liu et al., 2012). Hereby, we created the strategy to study the function of FGF5s in fine wool sheep by ectopic expression of FGF5s via lentivirus-mediated transgenesis. Of note, the wool length and fleece weight of transgenic sheep with ectopic expression of FGF5s were increased significantly comparing to wild type control. Our results strongly suggested that overexpression of FGF5s could stimulate wool growth and result in increase of wool length and weight. It potentiates the utility of FGF5s in improvement of wool length and productivity in the future.
Recombinant lentiviral particles were produced essentially as previously described (Klages et al., 2000). 293 T cells were seeded and cotransfected with pLEX-FGF5s (12 mg) and packaging plasmids (3.5 mg pMD2.G and 6 mg pPAX2) using Lipofectamine 2000 (Invitrogen). After 48-hour transfection, the supernatant containing lentivirus particles was filtered through 0.45-mm syringe filter and then concentrated by ultracentrifugation (Beckman) at 35,000 g for 3 h at 4 °C. Transgenic sheep were generated via injecting lentiviral particles into the perivitelline space of the zygotes (Liu et al., 2012). In brief, Sheep zygotes were collected from Xinjiang Merino Sheep. For lentivirus injection, around 50–100 pl of concentrated lentivirus with 3 × 109 IU/ml titer were injected into perivitelline space of zygotes using a micromanipulator. The zygotes were cultured in in vitro culture medium for 24 h until they divided into 2–4 cells. For embryo transfer, recipients were synchronized by the same treatment as donor ewes. Embryos injected with lentivirus were transferred to recipient ewes with mid-line laparotomy under general procedure.
2.4. Identification of the transgene integration Transgene integration was screened out by PCR with template of genomic DNA extracted from lamb tail using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). The primers spanning CMV promoter and FGF5s codon region were as followed: sense 5′-CACCAAAATCAACGGGACTT-3′ and anti-sense 5′GATGTTGCCGTCCTCCTTGAAGT-3′. PCR was performed with 300 ng genomic DNA in the condition of 95 °C denaturation for 2 min followed by 35 cycles of 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 60 s, ended at a final extension at 72 °C for 7 min. In order to further identify the transgene integration, southern blotting analysis was performed to determine copy numbers of transgene as previously described (Liu et al., 2012). In brief, genomic DNA samples were extracted from skin tissue and digested with EcoRI. After precipitation with alcohol, 10 mg digested DNA was separated and further transferred to nylon membrane. The 430 bp fragments of the CMV promoter were amplified as probe from pLEX-FGF5s construction using primers: sense 5′-CGAGGGCGATGCCACCTAC-3′ and antisense 5′CTCCAGC AGGACCATGTGATC-3′. The probe was prepared by 32PdCTP labeling with random primer extension kit (Promega) and hybridized with blotting membrane by incubating overnight at 65 °C in hybridization oven (Hoefer Scientific Instrument). Membranes were washed three times and exposed against film in dark cassette for 24 h. Then the film was developed as general protocol.
2. Materials and methods 2.1. Animals The animals used in this study were regularly maintained in the Research Base of Sheep Breeding of Xinjiang Academy of Animal Science, Xinjiang, China. Surgeries were performed under strict aseptic conditions, and efforts were made to minimize animal suffering. All animal handling procedures were carried out in strict accordance with approved guidelines of the Institutional Animal Care of Xinjiang Academy of Animal Science.
2.5. mRNA expression by RT-PCR Tissue samples were collected from the skin of transgenic sheep and normal sheep, and frozen immediately in liquid nitrogen for RNA extraction. Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and digested by DNase (Qiagen, Valencia, CA). First strand cDNA synthesis was performed using 1 μg of total RNA and reverse transcriptase, according to the manufacturer's protocol. Semi-quantitative RT-PCR was performed using a pair of primers (sense 5′-CTTGGAGCAGAGCAGCTTCCAGTGGAGC-3′ and anti-sense 5′-CCCATACGACGTCCCAGACTACG-3′) to detect the expression of the FGF5s in skin tissues of transgenic sheep and normal sheep. The β-actin mRNA as internal control was also amplified used the primers: sense 5′-CACGGCATCGTCACCAACTG-3′ and anti-sense 5′-CAGGGGTGTT GAAGGTCTCGAAC-3′. The mRNA abundance was estimated based on the amount of PCR products by agarose gel electrophoresis.
2.2. Construction of FGF5s expressing vector The ORF of ovine FGF5s (Genbank accession JQ9411957) derived from Merino sheep was previously described (Lihua et al., 2015). The restriction enzyme sites were engineered into the primer to facilitate subsequent fusion with the lentiviral vector. The FGF5s fragments were amplified by PCR using the primer pair 5′-ATAGCGGCCGCG(Not I)GAAGCATGAGCTT GTCCTT-3′ (sense) and 5′-GGCCTCGAG(XhoI) TCAagcgtagtctggg acgtcgtatgggta (HA tag) TCTGTAAATTTGGCTTAA C3′(anti-sense). Restriction sites were underlined and the sequence for the HA tag was incorporated into the reverse primer indicated in small letter. Then the FGF5s-HA was subcloned into the pLEX-mcs vector (Open Biosystem, Huntsville, AL, USA) followed by digesting with NotI and XhoI, named as pLEX-FGF5s. 478
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detected through common HA antibodies. The lentiviral expression construct was designed as pLEX-FGF5s (Fig. 1A). The sequence coding ovine FGF5s was cloned through reverse transcription PCR from cDNA derived from skin tissue of Merino sheep and the target fragments containing 378 bp FGF5s and 27 bp HA tag were carefully described in our previous study (Lihua et al., 2015). The expression of the recombinant FGF5s cassette in 293 T cells verified by western blotting clearly revealed that the size of recombinant FGF5s proteins was consistent with the expected, designed as 14.7 KD. The construct of pLEXFGF5s was then used to produce the transgenic sheep. We generated transgenic sheep with pLEX-FGF5s through injection of lentiviral particles into perivitelline space. A total of 63 one or two cell stage embryos were subjected to injection of recombinant lentivirus and subsequently transferred to 43 hormonally synchronized surrogate ewes. 18 surrogates were completed full term concept and produced 23 offsprings. By PCR screening, 20 transgenic lambs were identified. The tansgenic efficiency was as high as 87% (Fig. 1B & C). The results displayed that the lentiviral transgene delivery system worked with high efficiency to produce transgenic sheep with integration of recombinant FGF5s.
2.6. Western blotting Total proteins were extracted from skin tissues as previously described (Tian et al., 2013). Frozen samples were ground to powder and solubilized in the buffer of 62.5 mM Tris pH 6.8, 10% glycerol, 2.5% sodium dodecyl sulfate, and HaltTM-Protease Inhibitor Cocktail (Thermo Scientific). Quantification of total protein was carried out by Bicinchoninic acid assay with BSA (Sitransgenica-Aldrich). The proteins (100 mg) were subjected to 12% SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and immune-blotted with anti-HA (1:1000, TianGen) or anti-b-actin antibodies (1:10,000, Abcam). Immuno-reactive proteins were visualized using the Odyssey Infrared Imaging System and relatively quantified by densitometric analysis (LiCor, Lincoln, NE), according to the manufacturer's instructions. 2.7. Collection wool and measurement of phenotype Wool samples for determining the staple length and fiber mean diameter were removed from sheep of scapular region of body side (around the first rib area) once the transgenic sheep grow up to one year old. Total of 10 g for each sample were required for subsequent measurement of the staple length and fiber mean diameter. The staple length and wool greasy weight were manually measured followed by the national sheep wool quality Inspection standards of China (GB15232013). Fiber mean diameter was determined by OFDA 2000, according to GB/T21030-2007 instruction.
3.2. Determination of the transgene integration To determine recombinant FGF5s integration, PCR screening and southern blot assay were performed with genomic DNA derived from all the transgenic sheep. Using sense primer resided at CMV promoter area and anti-sense primer resided at recombinant FGF5s coding sequence, 638 bp amplicons spanning CMV promoter and FGF5s sequence were amplified in transgenic sheep (Fig. 2A). The target fragments were not observed in negative controls, indicating that the PCR products were specific for the recombinant CMV-FGF5s cassette. When the genomic DNA samples of transgenic sheep were digested by EcoRI and further hybridized with probe which is amplified from CMV promoter sequence of pLEX-FGF5s, the clear evidence for the presence and number of recombinant FGF5s cassette in transgenic sheep were observed and the number of recombinant integrants were ranged from 2 to 11 copies and for most individuals with 2 to 3 copies (Fig. 2B). Our results indicated that the recombinant FGF5s was integrated into multiple loci in transgenic sheep genome.
2.8. Statistical method The unpaired Student's t-test by SPSS 15.0 was performed where necessary. p < 0.05 was considered to be statistically significant. 3. Results 3.1. Generation of FGF5s transgenic sheep To examine the role of FGF5s in wool growth in merino sheep, we set out to produce transgenic sheep with ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. Given commercial antibodies against ovine FGF5s were limited, the sequence of HA tag was engineered into the antisense primer along with the restriction enzyme sites and recombinant FGF5s-HA proteins properly expressed would be
3.3. Analysis of FGF5s expression in transgenic lambs We next examined expression of recombinant FGF5s cassette in transgenic sheep. Using RT-PCR, we detected mRNA expression of recombinant Fig. 1. Generation of ectopic expression FGF5s transgenic sheep. A, Structures of FGF5s expression vector. The lentiviral vector pLEX was used to integrate the transgene of FGF5s-HA cassette. B, Transgenic sheep photos. Left, male transgenic sheep. Right, female transgenic sheep. C, Summary of generation of FGF5s transgenic sheep by highly efficient lentivirus-medicated transgene delivery system.
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Fig. 2. Identification of transgene integration in transgenic sheep. A, PCR-based amplification of FGF5s transgene from genomic DNA extracted from tail tips of newborn lambs. Amplicons are 638 bp fragments spanning CMV promoter and FGF5s sequences. M, DNA marker; pLEX-FGF5s vector, positive control. B, Southern blotting analysis of transgene integration of transgenic sheep. pLEX-FGF5s vector, positive control; NTC, non-transgenic sheep DNA used as negative control.
FGF5s transgenic lambs
Fig. 3. Determination of recombinant FGF5s expression in transgenic lambs. A, qRT-PCR assay. mRNA expression of recombinant FGF5s in skin tissue of transgenic lambs was detected using reverse transcription polymerase chain reaction. The numbering indicates RNA samples derived from transgenic lambs (left) and non-transgenic lambs (right), and the β-actin gene was used as an endogenous normalization control. B, Western Blot assay. Proteins extracted from skin tissue of eight transgenic lambs were subjected to immunoblotting with HA antibody (upper). β-Actin was used as loading control (lower).
Non-transgenic lambs
FGF5s-HA
-actin
14.7KDa
FGF5s-HA
-actin
43KDa
evaluation. The mean wool staple length of transgenic sheep carrying recombinant FGF5s gene and control group was 9.17 cm and 7.58 cm, respectively. The staple length of transgenic sheep was significantly longer than that of non-transgenic control (p < 0.01) (Fig. 4A;Table 1). It demonstrated that ectopic expression of FGF5s stimulated the wool growth and raised the wool length. Concordantly, the greasy fleece weight of transgenic sheep was significantly heavier than that of non-transgenic control, 3.22 kg of transgenic sheep versus 2.17 kg of control (p < 0.01) (Fig. 4B; Table 1). Notably, the mean fiber diameter of two groups showed no significant difference, with 17.40 μm of transgenic sheep versus 16.66 μm of control (Fig. 4C; Table 1). It indicated that the increase of wool weight most likely attributed to the increase of wool length. Moreover, even though the birth weight didn't show significant difference, the yearling body weight of transgenic sheep was significantly greater than that of wild type control animals (p < 0.01) (Fig. 4D; Table 1). Currently, we do not have a plausible explanation for the increase of yearling body weight in FGF5s transgenic sheep.
FGF5s in skin tissue of transgenic lambs. To distinguish expression of the endogenous FGF5s gene from that of the recombinant FGF5s cassette, antisense primer was designed to set within HA tag sequence. Among 12 live lambs, the expression of recombinant FGF5s were observed in all the transgenic sheep, and 11 animals robustly expressed recombinant FGF5s, while only one individual relatively weakly expressed the FGF5s (Fig. 3A). Subsequently, we determined the expression of recombinant FGF5s protein in transgenic lambs through Western Blotting. Proteins extracted from skin tissue of eight transgenic lambs were subjected to immunoblotting with antibodies against HA tag and clear evidence for the presence of recombinant proteins were observed (Fig. 3B). Our results demonstrated that recombinant FGF5s protein has been expressed in transgenic sheep.
3.4. Phenotype analysis of FGF5s transgenic sheep Except for 8 died lambs, the other 12 alive yearling transgenic founders and 12 wild-type control animals were subjected to phenotype 480
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P=0.001 P<0.001
P=0.001 P=0.540
Fig. 4. Evaluation of wool traits in FGF5s transgenic sheep and non-transgenic sheep. A, Wool staple length of yearling FGF5s transgenic sheep and non-transgenic sheep. Wool staple length were measured according to the national sheep wool quality Inspection standards of China (GB1523-2013). The average staple length of targeted lambs was significantly longer than those of control group. B, Yearling greasy fleece weight of FGF5s transgenic sheep and non-transgenic sheep. The average weight of targeted lamb was significant greater than that of control group. C, Yearling fiber mean diameter of FGF5s transgenic sheep and nontransgenic sheep. The fiber mean diameter was measured by OFDA 2000, according to GB/T21030-2007 instruction. D, Yearling weight of FGF5s transgenic sheep and non-transgenic sheep. The yearling weight of transgenic sheep was significantly greater than that of wide type control. Student's t-test was performed to determine the difference of wool trait between transgenic and non-transgenic sheep.
4. Discussion
with FGF5 for the binding to receptor FGFR1 (Suzuki et al., 2000). We isolated ovine FGF5 and its variant FGF5s from merino sheep (Lihua et al., 2015). The ovine FGF5 gene consists of three exons encoding a protein of 270 amino acid residues. The alternative splicing resulted in frame shifts and introduction of immediate stop codon in the third exon, therefore ovine FGF5s encodes a protein of 125 amino acid residues, of which the 120 amino acids in N-terminal are identical to that of FGF5. Hitherto, the function of FGF5 in fine wool sheep still remains unexplored. However, the high similarities between Merino sheep FGF5 gene sequence and other mammalian orthologs were observed in our previous study and it underlined that the role of FGF5 and its variant FGF5s in fine wool sheep could be similar to other animals. In this study, we used lentivirus-medicated transgene delivery system to create transgenic Chinese merino sheep overexpressed recombinant FGF5s. By western blotting, obvious evidences were provided to verify the high-level expression of the recombinant FGF5s cassette in skin tissue of transgenic merino sheep. Subsequent investigation on the wool traits of live lambs showed that the wool staple length and yearling grease fleece weight were significantly longer and heavier than those of wild-type non-transgenic animals, with 9.17 cm and 3.22 kg of transgenic sheep versus 7.58 cm and 2.17 kg of nontransgenic animals, respectively. These results indicated that ectopic expression of FGF5s promoted wool growth, and the effect was putatively reasoned to antagonizing endogenous FGF5 activity and relief of the inhibition of FGF5 in wool growth. Wool length is a critical economic trait in wool-producing sheep as it is closely associated to wool productivity and quality. The
In essence, wool growth is predominantly orchestrated by molecules that regulate fiber formation. The dermal papilla that resides within the base of the follicle acts as a control center for follicle activity. Continuous cell division in the follicle bulb region facilitates the movement of concentrically arranged inner root sheath, cuticles and cortical cells upwards toward the skin surface, accompanied by terminal differentiation of these cells. Thus, production of the harden wool fiber distinctly requires efficient activation of dermal papilla to satisfy constant cell division in follicle bulb region. FGF5 has been proved to be a potent candidate gene to regulate the hair growth. It is known that FGF5 blocks the activation of dermal papillae and consonantly induces the transition from hair growth to regression. Its biological activity is carried out through binding and activating the FGF receptor (Mohammadi et al., 2005). Meanwhile, its function can be regulated by alternative splicing. As a result of alternative mRNA splicing, FGF5 gene can produce a short form of the FGF5 protein as well as the full-length FGF5 protein, designated FGF5s and FGF5 respectively (Ozawa et al., 1998; Ito et al., 2003). FGF5 gene consists of three exons, whereas FGF5s lacks the sequence of whole exon 2 and part of the exon 3. FGF5s, a truncated form of FGF5, was firstly detected in rat (Hattori et al., 1996) and then it was identified in human (Ozawa et al., 1998), sheep (Lihua et al., 2015) and goat (Bao et al., 2015) in succession. Previous findings indicated that the truncated protein of FGF5s would repeal the FGF5 inhibition of hair growth. Moreover, FGF5s suppresses intracellular signaling through competing 481
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Table 1 Data of measurement for wool traits in yearling ectopic expression FGF5s transgenic sheep and non-transgenic sheep.
Transgenic sheep
Non-transgenic sheep
No.
Gender
Wool staple length (cm)
Yearling grease weight (kg)
Fiber diameter (μm)
Yearling body weight (kg)
110 115 116 117 118 122 129 130 131 135 144 145 Mean 104 109 113 120 127 147 911 912 913 914 916 917 Mean
♂ ♂ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♂
8.5 10.5 9.5 8.5 10.5 9.5 8.5 9 9.5 7.5 10 8.5 9.17 ± 0.91a 8.5 7.5 7.5 7 8 7.5 7.5 7.5 9 8 5 8 7.58 ± 0.97b
2.80 2.76 2.38 5.32 4.08 3.36 2.38 3.40 3.40 2.50 3.58 2.72 3.22 ± 0.85 1.50 1.88 2.00 2.64 2.90 1.94 2.10 2.66 2.16 2.44 1.80 2.12 2.17 ± 0.41
16.02 18.84 15.92 18.92 18.63 17.89 17.21 17.05 17.79 15.71 18.72 16.09 17.40 ± 1.24 a 16.53 17.86 16.82 20.71 15.64 14.54 14.34 14.9 17.27 19.49 18.15 13.69 16.67 ± 2.16a
38.5 39.5 31.16 50.48 44.44 36.5 32.7 39.96 37.68 30.1 40.5 34 37.96 ± 5.76 26.62 29.5 34.02 37.66 37.12 28.16 23.8 32.94 29.5 37.34 30.1 25.96 31.06 ± 4.72
♂ ♀ ♂ ♀ ♂ ♂ ♀ ♀ ♀ ♂ ♀ ♂
a
b
a
b
Note. Different letters indicated the significant level p < 0.001.
Science Foundation of China, a subcontract of grant 2013AA102506 from China National High Technology Research Development Program (863 Program).
improvement of wool length by conventional genetic means has been slow and not obvious. Among various approaches to abolish FGF5 function to promote hair growth, FGF5s is recognized to be promising substances against inhibition of hair growth. In mice, FGF5s is abundantly expressed in the hair follicles only during the latter anagen, suggesting that the two FGF5 gene products regulate hair growth in concert, presenting that FGF5 induces repression of hair growth and FGF5s antagonizes this activity during hair growth phase. Treatment of 10-amino acid partial FGF5 polypeptide aligning the receptor binding sites together with FGF5 into depilated mice skin resulted in faster hair growth and longer hair follicles than that in mice skin treated by FGF-5 alone (Ito et al., 2003). The involvement of FGF5s in hair growth was also addressed in cashmere goats. It was clearly displayed that FGF5s can regulate the activity of FGF5 at the level of its alternative splicing in cashmere goats. These findings are analogous to our results in Merino sheep, indicating that overexpression of FGF5s could stimulate wool growth and the utility of FGF5s in improvement of wool length and productivity would be feasible.
References Amendola, M., Venneri, M.A., Biffi, A., Vigna, E., Naldini, L., 2005. Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat. Biotechnol. 23 (1), 108. Bao, W.L., Yao, R.Y., He, Q., Guo, Z.X., Bao, C., Wang, Y.F., Wang, Z.G., 2015. Cloning, molecular characterization, and expression pattern of FGF5 in Cashmere goat (Capra hircus). Genet. Mol. Res. 14, 11154–11161. Dierks, C., Mömke, S., Philipp, U., Distl, O., 2013. Allelic heterogeneity of FGF5 mutations causes the long-hair phenotype in dogs. Anim. Genet. 44, 425–431. Drögemüller, C., Rüfenacht, S., Wichert, B., Leeb, T., 2007. Mutations within the FGF5 gene are associated with hair length in cats. Anim. Genet. 38, 218–221. Hattori, Y., Yamasaki, M., Itoh, N., 1996. The rat FGF-5 mRNA variant generated by alternative splicing encodes a novel truncated form of FGF-5. Biochim. Biophys. Acta 1306, 31–33. He, X., Yuan, C., Zhou, G., Chen, Y., 2015. Fibroblast growth factor 5-short (FGF5s) inhibits the activity of FGF5 in primary and secondary hair follicle dermal papilla cells of cashmere goats. Gene 575, 393–398. Hébert, J.M., Rosenquist, T., Götz, J., Martin, G.R., 1994. FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations. Cell 78, 1017–1025. Higgins, C.A., Petukhova, L., Harel, S., Ho, Y.Y., Drill, E., Shapiro, L., Wajid, M., Christiano, A.M., 2014. FGF5 is a crucial regulator of hair length in humans. Proc. Natl. Acad. Sci. 111, 10648–10653. Hofmann, A., Kessler, B., Ewerling, S., Weppert, M., Vogg, B., Ludwig, H., Stojkovic, M., Boelhauve, M., Brem, G., Wolf, E., Pfeifer, A., 2003. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep. 4, 1054–1060. Housley, D.J., Venta, P.J., 2006. The long and the short of it: evidence that FGF5 is a major determinant of canine 'hair'-itability. Anim. Genet. 37, 309–315. Ito, C., Saitoh, Y., Fujita, Y., Yamazaki, Y., Imamura, T., Oka, S., Suzukie, S., 2003. Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5. J. Cell. Physiol. 197, 272–283. Kijas, J.W., Lenstra, J.A., Hayes, B., Boitard, S., Porto Neto, L.R., San Cristobal, M., et al., 2012. Genome-wide analysis of the world's sheep breeds reveals high levels of historic mixture and strong recent selection. PLoS Biol. 10 (2), e1001258. Klages, N., Zufferey, R., Trono, D., 2000. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol. Ther. 2, 170–176. Konyukhov, B.V., Berdaliev, A.S., 1990. Analysis of the angora-Y gene expression. Mouse Genome 87, 94–95. Legrand, R., Tiret, L., Abitbol, M., 2014. Two recessive mutations in FGF5 are associated with the long-hair phenotype in donkeys. Genet. Sel. Evol. 46, 65–71. Lihua, Z., Sangang, H., Mingjun, L., Guosong, L., Zheng, Y., Chenxi, L., Xumei, Z., Ning,
5. Conclusions Fibroblast growth factor 5 (FGF5) has been recognized as an inhibitor to abolish hair growth, whereas FGF5 short alternative transcript (FGF5s) can induce hair growth by antagonizing FGF5 function. Hereby, we generated transgenic merino sheep with ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. The wool length and fleece weight of transgenic sheep were significantly greater than those of non-transgenic control. Our results strongly suggested that overexpression of FGF5s could stimulate wool growth and resulted in increase of wool length and greasy wool weight. Acknowledgement This study was funded by grant 2016ZX08008001-002-001 from China Agriculture Ministry genetically modified organisms project. It was also in part supported by grant U1303284 from National Natural 482
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Z., Wenrong, L., 2015. Molecular cloning, characterization, and expression of sheep FGF5 gene. Gene 555, 95–100. Liu, C.X., Wang, L.Q., L., W.R., Zhang, X.M., Tian, Y.Z., Zhang, N., He, S.G., Chen, T., Huang, J.C., Liu, M.J., 2012. Highly efficient generation of transgenic sheep by lentivirus accompanying the alteration of methylation status. PloS One 8, e54614. Mizuno, S., Iijima, S., Okano, T., Kajiwara, N., Kunita, S., Sugiyama, F., Yagami, K., 2011. Retrotransposon-mediated Fgf5(go-Utr) mutant mice with long pelage hair. Exp. Anim. 60, 161–167. Mohammadi, M., Olsen, S.K., Ibrahimi, O.A., 2005. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 16, 107–137. Nesterova, A., Nizamutdinov, I., Golovatenkoabramov, P., Konyukhov, B., 2010. Fluctuations of BMP signaling pathway during hair cycles in skin of mice with mutant genes we, wal and Fgf5(go). J. Dermatol. Sci. 60, 201–203. Ota, Y., Saitoh, Y., Suzuki, S., Ozawa, K., Kawano, M., Imamura, T., 2002. Fibroblast growth factor 5 inhibits hair growth by blocking dermal papilla cell activation.
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Research paper
Ectopic expression of FGF5s induces wool growth in Chinese merino sheep a,b,c
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MARK
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Wen-Rong Li , San-Gang He , Chen-Xi Liu , Xue-Mei Zhang , Li-Qin Wang , Jia-Peng Linb,c, Lei Chenb,c, Bin Hanb,c, Jun-Cheng Huangb,c, Ming-Jun Liub,c,⁎ a
College of Life Science and Technology, Xinjiang University, No. 14 Shengli road, Urmuqi, Xinjiang, China Key Laboratory of Genetics, Breeding & Reproduction of Grass-Feeding Livestock, Ministry of Agriculture, No. 468 Alishan road, Urmuqi, Xinjiang, China Key Laboratory of Animal Biotechnology of Xinjiang Institute of Animal Biotechnology, Xinjiang Academy of Animal Science, No. 468 Alishan road, Urmuqi, Xinjiang, China
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A B S T R A C T Fibroblast growth factor 5 (FGF5) has been recognized as an inhibitor to cease animal hair growth, while in contrary, FGF5 short alternative transcript (FGF5s) can induce hair growth by antagonizing FGF5 function. To investigate the role of FGF5s in wool growth in Chinese Merino sheep, we generated transgenic sheep of ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. Totally 20 transgenic sheep were obtained and 12 were alive after birth. Characterization of the transgene revealed that the transgenic sheep showed variety of integrant, ranged from 2 to 11 copies of transgene. The ectopic expression of FGF5s was observed in all transgenic sheep. Further study on the effect of ectopic expression of FGF5s revealed that the wool length of transgenic sheep were significantly longer than that of non-transgenic control, with 9.17 cm of transgenic lambs versus 7.58 cm of control animals. Notably, besides the increase of wool length, the yearling greasy fleece weight was also concordantly greater than that of wild-type (p < 0.01), with 3.22 kg of transgenic sheep versus 2.17 kg of control lambs (p < 0.01) in average. Our results suggested that overexpression of FGF5s could stimulate wool growth and resulted in increase of wool length and greasy wool weight.
1. Introduction Fine wool sheep had played critical role in textile and cloth industry in mankind history. Wool productivity and quality are essential factors for improvement of the profit of fine wool production. As a critical economic trait, wool fiber length is closely associated to wool productivity and quality. Under the similar wool density and fineness, the wool length has positive correlation to wool yield. Additionally, wool length is the secondly important quality trait coming after the fineness in wool textile industry, which influences the textile product quality and determines the manufacture system. Therefore, a considerable effort has been strengthened to increase the wool length by traditional genetic approaches in fine wool sheep breeding. In mice, a recessive phenotype known as angora(go), is featured by approximately 50% longer hair than wild type. The insight into the mechanism of “go” phenotype revealed that the go mice had longer anagen VI phase. It is the abnormal long anagen VI phase that results in the long hair phenotype (Konyukhov and Berdaliev, 1990). The subsequent research identified a null mutation of FGF5 gene which
contributed to the angora phenotype, and indicated that the transition between anagen and catagen is normally regulated by FGF5 (Hébert et al., 1994). Thereafter, by utility of state-of-art genomic approaches, substantial causal mutations in various species have been found to be related to hair length. Interestingly, all testified or candidate genes responsible for hair length were attributed to FGF5. So far, a number of spontaneous loss-of-function mutations of FGF5 associated to hair length have been identified in human (Higgins et al., 2014), mice (Nesterova et al., 2010; Mizuno et al., 2011), dogs ((Housley and Venta, 2006; Dierks et al., 2013), cats (Drögemüller et al., 2007)) and donkeys (Legrand et al., 2014). It is evidenced that FGF5 is the leading candidate gene for hair length variation. In sheep, FGF5 gene has been under intense selection (Kijas et al., 2012). However, no causal mutations have yet been identified to associate with wool length in fine wool sheep. FGF5 is a member of FGF family with 23 related genes and the role of FGF5 in hair growth is recognized as inhibitor to anagen phase. Sheep FGF5 was consisted by 3 exons and 2 introns spanned 21,743 bp genomic sequence (Lihua et al., 2015). By alternative splicing, FGF5
Abbreviations: FGF5, The fibroblast growth factor 5 gene; FGF5s, Fibroblast growth factor 5-short; FGF, The fibroblast growth factor; FGFR1, The fibroblast growth factor receptor1; FGFR2, The fibroblast growth factor receptor2 ⁎ Corresponding author at: Key Laboratory of Genetics, Breeding & Reproduction of Grass-Feeding Livestock, Ministry of Agriculture, No. 468 Alishan road, Urmuqi, Xinjiang, China. E-mail address: [email protected] (M.-J. Liu). http://dx.doi.org/10.1016/j.gene.2017.06.037 Received 5 May 2017; Received in revised form 15 June 2017; Accepted 20 June 2017 Available online 28 June 2017 0378-1119/ © 2017 Published by Elsevier B.V.
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2.3. Preparation of lentiviral particles and generation of transgenic sheep
gene produces two transcripts, the full length FGF5 with complete three exons and short alternative transcript (FGF5s) without exon2 and part of exon3. The full length transcript which functions to inhibit the activation of derma papilla cells proliferation and synthesis of hair fiber during anagen, and promotes the transition from anagen to catagen (Ota et al., 2002). The FGF5s has no inhibitory function in hair growth, whereas it binds to the FGF receptor1 (FGFR1) and 2 (FGFR2) to compete the FGF5 binding and serves as an antagonist to inhibit FGF5 function (Ozawa et al., 1998). Previous studies addressed that FGF5s could relieve hair growth inhibition in mice and cashmere goats(Suzuki et al., 2000; He et al., 2015) In contrast to other animals, the wool growth in fine wool sheep is distinct by life-long growing without seasonal spontaneous hair removal. So the function of FGF5s in fine wool sheep is still far from understood. Given that the high similarity between Merino sheep FGF5 gene sequence and other mammalian orthologs was observed in our previous study (Lihua et al., 2015), we postulated that the wide type FGF5 could be likely to function inhibitory effect in fine wool sheep, and FGF5s could serve as its antagonist to stimulate wool growth. Nowadays, with the fast development of genome modification approaches, scientists have applied the versatile gene manipulating approaches to modify genes associated with specific traits. Knockout or disruption of FGF5 by gene targeting in mice or cashmere goats resulted in increase of hair or cashmere length (Hébert et al., 1994; Wang et al., 2016). Lentiviral transgene delivery system offers high efficient means in producing transgenic animals (Hofmann et al., 2003; Pfeifer, 2004; Amendola et al., 2005). Our previous study documented that lentiviral transgenesis achieved high efficiency and widespread expression of EGFP in transgenic sheep (Liu et al., 2012). Hereby, we created the strategy to study the function of FGF5s in fine wool sheep by ectopic expression of FGF5s via lentivirus-mediated transgenesis. Of note, the wool length and fleece weight of transgenic sheep with ectopic expression of FGF5s were increased significantly comparing to wild type control. Our results strongly suggested that overexpression of FGF5s could stimulate wool growth and result in increase of wool length and weight. It potentiates the utility of FGF5s in improvement of wool length and productivity in the future.
Recombinant lentiviral particles were produced essentially as previously described (Klages et al., 2000). 293 T cells were seeded and cotransfected with pLEX-FGF5s (12 mg) and packaging plasmids (3.5 mg pMD2.G and 6 mg pPAX2) using Lipofectamine 2000 (Invitrogen). After 48-hour transfection, the supernatant containing lentivirus particles was filtered through 0.45-mm syringe filter and then concentrated by ultracentrifugation (Beckman) at 35,000 g for 3 h at 4 °C. Transgenic sheep were generated via injecting lentiviral particles into the perivitelline space of the zygotes (Liu et al., 2012). In brief, Sheep zygotes were collected from Xinjiang Merino Sheep. For lentivirus injection, around 50–100 pl of concentrated lentivirus with 3 × 109 IU/ml titer were injected into perivitelline space of zygotes using a micromanipulator. The zygotes were cultured in in vitro culture medium for 24 h until they divided into 2–4 cells. For embryo transfer, recipients were synchronized by the same treatment as donor ewes. Embryos injected with lentivirus were transferred to recipient ewes with mid-line laparotomy under general procedure.
2.4. Identification of the transgene integration Transgene integration was screened out by PCR with template of genomic DNA extracted from lamb tail using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). The primers spanning CMV promoter and FGF5s codon region were as followed: sense 5′-CACCAAAATCAACGGGACTT-3′ and anti-sense 5′GATGTTGCCGTCCTCCTTGAAGT-3′. PCR was performed with 300 ng genomic DNA in the condition of 95 °C denaturation for 2 min followed by 35 cycles of 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 60 s, ended at a final extension at 72 °C for 7 min. In order to further identify the transgene integration, southern blotting analysis was performed to determine copy numbers of transgene as previously described (Liu et al., 2012). In brief, genomic DNA samples were extracted from skin tissue and digested with EcoRI. After precipitation with alcohol, 10 mg digested DNA was separated and further transferred to nylon membrane. The 430 bp fragments of the CMV promoter were amplified as probe from pLEX-FGF5s construction using primers: sense 5′-CGAGGGCGATGCCACCTAC-3′ and antisense 5′CTCCAGC AGGACCATGTGATC-3′. The probe was prepared by 32PdCTP labeling with random primer extension kit (Promega) and hybridized with blotting membrane by incubating overnight at 65 °C in hybridization oven (Hoefer Scientific Instrument). Membranes were washed three times and exposed against film in dark cassette for 24 h. Then the film was developed as general protocol.
2. Materials and methods 2.1. Animals The animals used in this study were regularly maintained in the Research Base of Sheep Breeding of Xinjiang Academy of Animal Science, Xinjiang, China. Surgeries were performed under strict aseptic conditions, and efforts were made to minimize animal suffering. All animal handling procedures were carried out in strict accordance with approved guidelines of the Institutional Animal Care of Xinjiang Academy of Animal Science.
2.5. mRNA expression by RT-PCR Tissue samples were collected from the skin of transgenic sheep and normal sheep, and frozen immediately in liquid nitrogen for RNA extraction. Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and digested by DNase (Qiagen, Valencia, CA). First strand cDNA synthesis was performed using 1 μg of total RNA and reverse transcriptase, according to the manufacturer's protocol. Semi-quantitative RT-PCR was performed using a pair of primers (sense 5′-CTTGGAGCAGAGCAGCTTCCAGTGGAGC-3′ and anti-sense 5′-CCCATACGACGTCCCAGACTACG-3′) to detect the expression of the FGF5s in skin tissues of transgenic sheep and normal sheep. The β-actin mRNA as internal control was also amplified used the primers: sense 5′-CACGGCATCGTCACCAACTG-3′ and anti-sense 5′-CAGGGGTGTT GAAGGTCTCGAAC-3′. The mRNA abundance was estimated based on the amount of PCR products by agarose gel electrophoresis.
2.2. Construction of FGF5s expressing vector The ORF of ovine FGF5s (Genbank accession JQ9411957) derived from Merino sheep was previously described (Lihua et al., 2015). The restriction enzyme sites were engineered into the primer to facilitate subsequent fusion with the lentiviral vector. The FGF5s fragments were amplified by PCR using the primer pair 5′-ATAGCGGCCGCG(Not I)GAAGCATGAGCTT GTCCTT-3′ (sense) and 5′-GGCCTCGAG(XhoI) TCAagcgtagtctggg acgtcgtatgggta (HA tag) TCTGTAAATTTGGCTTAA C3′(anti-sense). Restriction sites were underlined and the sequence for the HA tag was incorporated into the reverse primer indicated in small letter. Then the FGF5s-HA was subcloned into the pLEX-mcs vector (Open Biosystem, Huntsville, AL, USA) followed by digesting with NotI and XhoI, named as pLEX-FGF5s. 478
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detected through common HA antibodies. The lentiviral expression construct was designed as pLEX-FGF5s (Fig. 1A). The sequence coding ovine FGF5s was cloned through reverse transcription PCR from cDNA derived from skin tissue of Merino sheep and the target fragments containing 378 bp FGF5s and 27 bp HA tag were carefully described in our previous study (Lihua et al., 2015). The expression of the recombinant FGF5s cassette in 293 T cells verified by western blotting clearly revealed that the size of recombinant FGF5s proteins was consistent with the expected, designed as 14.7 KD. The construct of pLEXFGF5s was then used to produce the transgenic sheep. We generated transgenic sheep with pLEX-FGF5s through injection of lentiviral particles into perivitelline space. A total of 63 one or two cell stage embryos were subjected to injection of recombinant lentivirus and subsequently transferred to 43 hormonally synchronized surrogate ewes. 18 surrogates were completed full term concept and produced 23 offsprings. By PCR screening, 20 transgenic lambs were identified. The tansgenic efficiency was as high as 87% (Fig. 1B & C). The results displayed that the lentiviral transgene delivery system worked with high efficiency to produce transgenic sheep with integration of recombinant FGF5s.
2.6. Western blotting Total proteins were extracted from skin tissues as previously described (Tian et al., 2013). Frozen samples were ground to powder and solubilized in the buffer of 62.5 mM Tris pH 6.8, 10% glycerol, 2.5% sodium dodecyl sulfate, and HaltTM-Protease Inhibitor Cocktail (Thermo Scientific). Quantification of total protein was carried out by Bicinchoninic acid assay with BSA (Sitransgenica-Aldrich). The proteins (100 mg) were subjected to 12% SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) and immune-blotted with anti-HA (1:1000, TianGen) or anti-b-actin antibodies (1:10,000, Abcam). Immuno-reactive proteins were visualized using the Odyssey Infrared Imaging System and relatively quantified by densitometric analysis (LiCor, Lincoln, NE), according to the manufacturer's instructions. 2.7. Collection wool and measurement of phenotype Wool samples for determining the staple length and fiber mean diameter were removed from sheep of scapular region of body side (around the first rib area) once the transgenic sheep grow up to one year old. Total of 10 g for each sample were required for subsequent measurement of the staple length and fiber mean diameter. The staple length and wool greasy weight were manually measured followed by the national sheep wool quality Inspection standards of China (GB15232013). Fiber mean diameter was determined by OFDA 2000, according to GB/T21030-2007 instruction.
3.2. Determination of the transgene integration To determine recombinant FGF5s integration, PCR screening and southern blot assay were performed with genomic DNA derived from all the transgenic sheep. Using sense primer resided at CMV promoter area and anti-sense primer resided at recombinant FGF5s coding sequence, 638 bp amplicons spanning CMV promoter and FGF5s sequence were amplified in transgenic sheep (Fig. 2A). The target fragments were not observed in negative controls, indicating that the PCR products were specific for the recombinant CMV-FGF5s cassette. When the genomic DNA samples of transgenic sheep were digested by EcoRI and further hybridized with probe which is amplified from CMV promoter sequence of pLEX-FGF5s, the clear evidence for the presence and number of recombinant FGF5s cassette in transgenic sheep were observed and the number of recombinant integrants were ranged from 2 to 11 copies and for most individuals with 2 to 3 copies (Fig. 2B). Our results indicated that the recombinant FGF5s was integrated into multiple loci in transgenic sheep genome.
2.8. Statistical method The unpaired Student's t-test by SPSS 15.0 was performed where necessary. p < 0.05 was considered to be statistically significant. 3. Results 3.1. Generation of FGF5s transgenic sheep To examine the role of FGF5s in wool growth in merino sheep, we set out to produce transgenic sheep with ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. Given commercial antibodies against ovine FGF5s were limited, the sequence of HA tag was engineered into the antisense primer along with the restriction enzyme sites and recombinant FGF5s-HA proteins properly expressed would be
3.3. Analysis of FGF5s expression in transgenic lambs We next examined expression of recombinant FGF5s cassette in transgenic sheep. Using RT-PCR, we detected mRNA expression of recombinant Fig. 1. Generation of ectopic expression FGF5s transgenic sheep. A, Structures of FGF5s expression vector. The lentiviral vector pLEX was used to integrate the transgene of FGF5s-HA cassette. B, Transgenic sheep photos. Left, male transgenic sheep. Right, female transgenic sheep. C, Summary of generation of FGF5s transgenic sheep by highly efficient lentivirus-medicated transgene delivery system.
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Fig. 2. Identification of transgene integration in transgenic sheep. A, PCR-based amplification of FGF5s transgene from genomic DNA extracted from tail tips of newborn lambs. Amplicons are 638 bp fragments spanning CMV promoter and FGF5s sequences. M, DNA marker; pLEX-FGF5s vector, positive control. B, Southern blotting analysis of transgene integration of transgenic sheep. pLEX-FGF5s vector, positive control; NTC, non-transgenic sheep DNA used as negative control.
FGF5s transgenic lambs
Fig. 3. Determination of recombinant FGF5s expression in transgenic lambs. A, qRT-PCR assay. mRNA expression of recombinant FGF5s in skin tissue of transgenic lambs was detected using reverse transcription polymerase chain reaction. The numbering indicates RNA samples derived from transgenic lambs (left) and non-transgenic lambs (right), and the β-actin gene was used as an endogenous normalization control. B, Western Blot assay. Proteins extracted from skin tissue of eight transgenic lambs were subjected to immunoblotting with HA antibody (upper). β-Actin was used as loading control (lower).
Non-transgenic lambs
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-actin
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-actin
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evaluation. The mean wool staple length of transgenic sheep carrying recombinant FGF5s gene and control group was 9.17 cm and 7.58 cm, respectively. The staple length of transgenic sheep was significantly longer than that of non-transgenic control (p < 0.01) (Fig. 4A;Table 1). It demonstrated that ectopic expression of FGF5s stimulated the wool growth and raised the wool length. Concordantly, the greasy fleece weight of transgenic sheep was significantly heavier than that of non-transgenic control, 3.22 kg of transgenic sheep versus 2.17 kg of control (p < 0.01) (Fig. 4B; Table 1). Notably, the mean fiber diameter of two groups showed no significant difference, with 17.40 μm of transgenic sheep versus 16.66 μm of control (Fig. 4C; Table 1). It indicated that the increase of wool weight most likely attributed to the increase of wool length. Moreover, even though the birth weight didn't show significant difference, the yearling body weight of transgenic sheep was significantly greater than that of wild type control animals (p < 0.01) (Fig. 4D; Table 1). Currently, we do not have a plausible explanation for the increase of yearling body weight in FGF5s transgenic sheep.
FGF5s in skin tissue of transgenic lambs. To distinguish expression of the endogenous FGF5s gene from that of the recombinant FGF5s cassette, antisense primer was designed to set within HA tag sequence. Among 12 live lambs, the expression of recombinant FGF5s were observed in all the transgenic sheep, and 11 animals robustly expressed recombinant FGF5s, while only one individual relatively weakly expressed the FGF5s (Fig. 3A). Subsequently, we determined the expression of recombinant FGF5s protein in transgenic lambs through Western Blotting. Proteins extracted from skin tissue of eight transgenic lambs were subjected to immunoblotting with antibodies against HA tag and clear evidence for the presence of recombinant proteins were observed (Fig. 3B). Our results demonstrated that recombinant FGF5s protein has been expressed in transgenic sheep.
3.4. Phenotype analysis of FGF5s transgenic sheep Except for 8 died lambs, the other 12 alive yearling transgenic founders and 12 wild-type control animals were subjected to phenotype 480
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P=0.001 P<0.001
P=0.001 P=0.540
Fig. 4. Evaluation of wool traits in FGF5s transgenic sheep and non-transgenic sheep. A, Wool staple length of yearling FGF5s transgenic sheep and non-transgenic sheep. Wool staple length were measured according to the national sheep wool quality Inspection standards of China (GB1523-2013). The average staple length of targeted lambs was significantly longer than those of control group. B, Yearling greasy fleece weight of FGF5s transgenic sheep and non-transgenic sheep. The average weight of targeted lamb was significant greater than that of control group. C, Yearling fiber mean diameter of FGF5s transgenic sheep and nontransgenic sheep. The fiber mean diameter was measured by OFDA 2000, according to GB/T21030-2007 instruction. D, Yearling weight of FGF5s transgenic sheep and non-transgenic sheep. The yearling weight of transgenic sheep was significantly greater than that of wide type control. Student's t-test was performed to determine the difference of wool trait between transgenic and non-transgenic sheep.
4. Discussion
with FGF5 for the binding to receptor FGFR1 (Suzuki et al., 2000). We isolated ovine FGF5 and its variant FGF5s from merino sheep (Lihua et al., 2015). The ovine FGF5 gene consists of three exons encoding a protein of 270 amino acid residues. The alternative splicing resulted in frame shifts and introduction of immediate stop codon in the third exon, therefore ovine FGF5s encodes a protein of 125 amino acid residues, of which the 120 amino acids in N-terminal are identical to that of FGF5. Hitherto, the function of FGF5 in fine wool sheep still remains unexplored. However, the high similarities between Merino sheep FGF5 gene sequence and other mammalian orthologs were observed in our previous study and it underlined that the role of FGF5 and its variant FGF5s in fine wool sheep could be similar to other animals. In this study, we used lentivirus-medicated transgene delivery system to create transgenic Chinese merino sheep overexpressed recombinant FGF5s. By western blotting, obvious evidences were provided to verify the high-level expression of the recombinant FGF5s cassette in skin tissue of transgenic merino sheep. Subsequent investigation on the wool traits of live lambs showed that the wool staple length and yearling grease fleece weight were significantly longer and heavier than those of wild-type non-transgenic animals, with 9.17 cm and 3.22 kg of transgenic sheep versus 7.58 cm and 2.17 kg of nontransgenic animals, respectively. These results indicated that ectopic expression of FGF5s promoted wool growth, and the effect was putatively reasoned to antagonizing endogenous FGF5 activity and relief of the inhibition of FGF5 in wool growth. Wool length is a critical economic trait in wool-producing sheep as it is closely associated to wool productivity and quality. The
In essence, wool growth is predominantly orchestrated by molecules that regulate fiber formation. The dermal papilla that resides within the base of the follicle acts as a control center for follicle activity. Continuous cell division in the follicle bulb region facilitates the movement of concentrically arranged inner root sheath, cuticles and cortical cells upwards toward the skin surface, accompanied by terminal differentiation of these cells. Thus, production of the harden wool fiber distinctly requires efficient activation of dermal papilla to satisfy constant cell division in follicle bulb region. FGF5 has been proved to be a potent candidate gene to regulate the hair growth. It is known that FGF5 blocks the activation of dermal papillae and consonantly induces the transition from hair growth to regression. Its biological activity is carried out through binding and activating the FGF receptor (Mohammadi et al., 2005). Meanwhile, its function can be regulated by alternative splicing. As a result of alternative mRNA splicing, FGF5 gene can produce a short form of the FGF5 protein as well as the full-length FGF5 protein, designated FGF5s and FGF5 respectively (Ozawa et al., 1998; Ito et al., 2003). FGF5 gene consists of three exons, whereas FGF5s lacks the sequence of whole exon 2 and part of the exon 3. FGF5s, a truncated form of FGF5, was firstly detected in rat (Hattori et al., 1996) and then it was identified in human (Ozawa et al., 1998), sheep (Lihua et al., 2015) and goat (Bao et al., 2015) in succession. Previous findings indicated that the truncated protein of FGF5s would repeal the FGF5 inhibition of hair growth. Moreover, FGF5s suppresses intracellular signaling through competing 481
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Table 1 Data of measurement for wool traits in yearling ectopic expression FGF5s transgenic sheep and non-transgenic sheep.
Transgenic sheep
Non-transgenic sheep
No.
Gender
Wool staple length (cm)
Yearling grease weight (kg)
Fiber diameter (μm)
Yearling body weight (kg)
110 115 116 117 118 122 129 130 131 135 144 145 Mean 104 109 113 120 127 147 911 912 913 914 916 917 Mean
♂ ♂ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♂
8.5 10.5 9.5 8.5 10.5 9.5 8.5 9 9.5 7.5 10 8.5 9.17 ± 0.91a 8.5 7.5 7.5 7 8 7.5 7.5 7.5 9 8 5 8 7.58 ± 0.97b
2.80 2.76 2.38 5.32 4.08 3.36 2.38 3.40 3.40 2.50 3.58 2.72 3.22 ± 0.85 1.50 1.88 2.00 2.64 2.90 1.94 2.10 2.66 2.16 2.44 1.80 2.12 2.17 ± 0.41
16.02 18.84 15.92 18.92 18.63 17.89 17.21 17.05 17.79 15.71 18.72 16.09 17.40 ± 1.24 a 16.53 17.86 16.82 20.71 15.64 14.54 14.34 14.9 17.27 19.49 18.15 13.69 16.67 ± 2.16a
38.5 39.5 31.16 50.48 44.44 36.5 32.7 39.96 37.68 30.1 40.5 34 37.96 ± 5.76 26.62 29.5 34.02 37.66 37.12 28.16 23.8 32.94 29.5 37.34 30.1 25.96 31.06 ± 4.72
♂ ♀ ♂ ♀ ♂ ♂ ♀ ♀ ♀ ♂ ♀ ♂
a
b
a
b
Note. Different letters indicated the significant level p < 0.001.
Science Foundation of China, a subcontract of grant 2013AA102506 from China National High Technology Research Development Program (863 Program).
improvement of wool length by conventional genetic means has been slow and not obvious. Among various approaches to abolish FGF5 function to promote hair growth, FGF5s is recognized to be promising substances against inhibition of hair growth. In mice, FGF5s is abundantly expressed in the hair follicles only during the latter anagen, suggesting that the two FGF5 gene products regulate hair growth in concert, presenting that FGF5 induces repression of hair growth and FGF5s antagonizes this activity during hair growth phase. Treatment of 10-amino acid partial FGF5 polypeptide aligning the receptor binding sites together with FGF5 into depilated mice skin resulted in faster hair growth and longer hair follicles than that in mice skin treated by FGF-5 alone (Ito et al., 2003). The involvement of FGF5s in hair growth was also addressed in cashmere goats. It was clearly displayed that FGF5s can regulate the activity of FGF5 at the level of its alternative splicing in cashmere goats. These findings are analogous to our results in Merino sheep, indicating that overexpression of FGF5s could stimulate wool growth and the utility of FGF5s in improvement of wool length and productivity would be feasible.
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5. Conclusions Fibroblast growth factor 5 (FGF5) has been recognized as an inhibitor to abolish hair growth, whereas FGF5 short alternative transcript (FGF5s) can induce hair growth by antagonizing FGF5 function. Hereby, we generated transgenic merino sheep with ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. The wool length and fleece weight of transgenic sheep were significantly greater than those of non-transgenic control. Our results strongly suggested that overexpression of FGF5s could stimulate wool growth and resulted in increase of wool length and greasy wool weight. Acknowledgement This study was funded by grant 2016ZX08008001-002-001 from China Agriculture Ministry genetically modified organisms project. It was also in part supported by grant U1303284 from National Natural 482
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