Plasmid Based Expression and Bioactivity Evaluation of Caprine Growth Hormone Gene Cloned from a Local Pakistani Goat Breed Beetal.
Growth hormone cDNA of Beetal goat (Capra hircus) an indigenous breed of Punjab Pakistan was amplified by RT PCR and gene including leader sequence was cloned in pTZR57 cloning vector. The cGH-pTZR57 clone was confirmed by restriction digestion and sequence analyses before finally sub-cloning the gene in pND - a mammalian expression vector. The clones were again confirmed by restriction digestion and PCR analyses. Highly purified super coiled recombinant caprine growth hormone-pND (rcGH-pND) construct was used to transfect Vero cell lines for expression studies. The in vitro expression of cGH was determined by dot-ELISA. After confirming its in vitro cell line based expression rcGH-pN pND constructs and phosphate buffer saline (PBS) were separately injected to 4 weeks old balb/c female mice intramuscularly. Total forty five animals were used to determine the biological activity and randomly divided in to three groups with fifteen mice in each group.
Five animals from each group were used to monitor the in vivo biological activity by evaluating the body weight gain and tibia epiphyseal width assays from zero week up to four weeks with one week gap. Significant increase (P less than 0.05) gain in body weight and in tibia epiphyseal width was observed in animals inoculated with rcGH-pN. Thus it is concluded that recombinant plasmid of cGH cDNA may be used as supplement to increase the meat production. The effects of the same plasmid on milk production may be checked in future experiments using Beetal goat. Copyright 2014 Friends Science Publishers
Keywords: Caprine growth hormone; Mammalian expression vector pND; Vero cell line; Beetal growth hormone
The economy of Pakistan is mainly dependent upon agriculture and livestock is the second important sector of agriculture. Pakistan is rich in its livestock however due to urbanization and increasing population size demands for meat and milk is also increasing. Since past efforts are being made to cope with these demands by different conventional methods such as selection breeding improved management establishment of more dairy herds and utilization of various scientific techniques (Zhang et al. 2013). One of these methods is to strength existing and/or create new infrastructure to facilitate the milk supply along with the stress on milk production (Chattha et al. 2013). Beside all these conventional methods one of the latest scientific techniques is the use of recombinant technology for the production of valuable proteins like bovine growth hormone (bGH). It is a multifunctional naturally occurring non-
glycosylated protein hormone secreted from the anterior pituitary gland having 190 amino acids. Its biological effects are broadly classified as either somatogenic or metabolic which results in linear growth and milk production of the ruminant system (Pell and Bates 1987). The somatogenic effects are mediated by insulin like growth factor-1 (IGF-1) while the metabolic effect involves a variety of tissues and general metabolism of carbohydrate lipid protein and minerals essential for growth (Etherton and Bauman 1998). For different biological roles and commercial application in animal husbandry GH has been cloned from a number of species and expressed in various expression systems like prokaryotic (Wingfield et al. 1987; Mukhopadhyay and Sahni 2002; Khan et al. 2007 Khalid et al. 2008) and eukaryotic system (Hawkins and Nakamura 1999) to produce high levels of recombinant protein which is being commercially used for enhancement of milk and meat production in cattle (Baldi 1999; Bauman 1999).
Because of high genetic potentials and low productivity of milk and meat in most of South East Asian cattle and small ruminant breeds the exogenous administration of recombinant homologous GH approaches on one hand provide the key for boosting the productivity of ruminants to increase the food output (meat or milk) per unit of food source input. This is because of reduction in animal waste products and expenditures for animal feed production (Bauman 1992). On the other hand the state of the art plasmid based expression technology is getting popular in recent years (Patil et al. 2005). For example to screen the targets identified from genomic projects to shuffle molecules for vaccination or direct in vivo production of hormones and other therapeutics or preventive applications (Draghia-Akli et al. 2006).
This economically feasible gene delivery technology and plasmid mediated gene transfer is also emerging as an excellent candidate for agriculture applications to optimize production and animal welfare (Draghia-Akli and Fiorotto 2004; Cunningham 2005).
Goats (Capra hircus) are fastest growing ruminants in Pakistan and about 25 goat breeds are in the country and two wild relative such as Markhor and Ibex (Khan et al. 2008; Shahzad et al. 2012). These are important for poor livestock farmers to make a living and according to recent data over 64 million goats are available in Pakistan (GOP 2013). Some species are good for meat or milk production. For example Beetal goats are famous for its milk and meat production and is called poor man's cow (Iqbal et al. 2008; Qudus et al. 2013). These goats are found in almost all irrigated areas of the Punjab including districts of Jhelum Gujarat Mandi-Bahaudddin Sialkot Gujranwala Lahore Sheikhupura Faisalabad Sargodha Jhang Multan Sahiwal and Okara (www.haasil-foundation.com/).
Keeping in view the importance of Beetal breed's contribution towards meat and milk production this study was designed to clone the caprine growth hormone (cGH) cDNA as a supplement agent into a mammalian expression vector to carry out expression studies in vitro using animal cell line and then to evaluate its biological response in vivo using animal model. This may help to develop feasible plasmid based delivery system for growth hormone cloned from various farming animals to enhance the production of milk and meat in high demand market like South East Asian countries and help to raise livestock sector.
Materials and Methods
Caprine GH-pTZR57 construct in DH5a cells with accession # DQ 307368 by Khan et al. (2005) was available in Biotechnology Laboratory Department of Biochemistry PMAS-Arid Agriculture University Rawalpindi. The clone was refreshed from its glycerol stock and plasmid DNA was extracted by the method described by Maniatis et al. (1989). Similarly mammalian expression vector pND cloned in
DH5a cells was also refreshed by streaking and growing on Lauria-Bertani (LB) media prepared by the method mentioned by Maniatis et al. (1989). Plasmid DNA of pND vector was extracted to be used in sub-cloning experiment. This vector contains constitutive immediate early promoter and enhancer sequences from cytomegalovirus (CMV) and 1000 bp intron sequence for optimal gene expression under in vitro and in vivo conditions. Before starting experiments the protocols were reviewed and approved by the ethical committee of PMAS Arid Agriculture University Rawalpindi.
Sub-cloning of cGH in pND
The cGH-pTZR57 construct was restricted with Xba I and Bam HI to produce the 692 bp long cGH gene with leader sequence. Similarly pND vector was modified by restriction digestion with Nhe I and Bam HI enzymes. The fragments were purified from gel by gel extraction kit (Fermentas USA) and purified products were ligated with T4 ligase using standard ligation procedure provided by Supplier (Roche USA). The ligation mixture was then transformed into chemically competent B10 strain of E. coli. The positive clones were confirmed by restriction digestion using EcoRV and agarose gel electrophoresis.
DNA used for expression studies was prepared by Quantum prep Plasmid Midiprep kit (Bio-Rad USA). DNA concentration was determined by spectrophotometer at optical density of 260 nm and 280 nm. The ratio of 260/280 nm was used to determine the quality of DNA. Most of DNA samples gave ratio between 1.7 and 2.0. The samples with lower ratio were retreated with chloroform- isoamylalcohol to remove proteins. The revived Vero cells obtained from National Institute of Health (NIH) Islamabad were used for expression studies. The cells were grown in Medium 199-Hepes containing 10% fetal bovine serum 1% penicillin streptomycin solution (PSS) 1% amphotericin B and 0.6% L-glutamine by culturing the cells in 12 wells plate (Corning USA) and incubated in the humidified CO2 incubator at 37oC. Cells with 80% of confluence were used for transfection experiments using calcium phosphate co- precipitation method described by Wigler et al. (1978). After transfection the cells were incubated from 48 to 96 h.
Thereafter the media and cell monolayer were harvested and analyzed by dot blot.
The biological activity of the construct i.e. rcGH cDNA was evaluated in white BALB/c an albino female mice by weight gain and tibia epiphyseal width assays using endotoxin free plasmid DNAs (Greenspan et al. 1949; Zhu et al. 1997). Forty five four weeks old BALB/c mice were divided into three groups containing fifteen animals each and kept at animal house facility of National Institute of Health (NIH) Islamabad to acclimatize for one week before inoculating with plasmid DNAs. First group of mice were injected with 100 g pND vector without insert in100 L of phosphate buffer saline (PBS) second group was injected with 100 L of PBS solution and third group was injected with 100 g rcGH-pND in 100 L PBS of intramuscularly. All animals were examined for change in body weight and width of the tibial epiphysis starting from zero week till the end of four weeks with one week interval. The data was analyzed by using analysis of variance (ANOVA) through MS Excel Data analysis tool Version 2007.
The means with significant difference were compared with Duncan Multiple Range Test (DMRT) using M-STAT C.
The primary objective of this study was to evaluate the bioactivity of cloned GH cDNA from caprine breed; Beetal using novel DNA-based expression technology.
Sub-cloning of cGH cDNA in pND Vector
The sub cloning of cGH cDNA in pND vector was confirmed by restricting the clone with restriction enzyme; EcoR V which generated an expected fragment of 692 bp. The obtained result is shown in Fig. 1.
Expression of cGH cDNA was done in Vero cell line. The maximum level of expression was observed with 2.5 g of construct i.e. rcGH-pND at 72 h of post transfection in cultural media. The pND without insert i.e. rcGH was used as negative control while commercially available GH hormone was used as positive control in these in vitro expression studies which showed no expression and strong expression respectively (Fig. 2). Similarly level of expression determined in cell lysate showed no detectable expression at any incubation hours with both concentrations of rcGH-pND used i.e. 2.5 g and 5 g (Fig. 3).
The bioactivity evaluation of plasmid based recombinant caprine growth hormone (rcGH-pND) by weight gain and tibia width bioassays in BALB/c female mice showed that there was significant increase in the weight (P less than 0.05) as well as in width of tibia (P less than 0.05) of the mice from zero to fourth week of the experiment showing gradual normal growth pattern in comparison to PBS and pND control (Table 1 2).
Expression studies carried out by other investigators using GH cDNA from other species like human and ovine (which is identical to caprine and thus can be used in both animals; ovine and caprine) using different cell lines i.e. COS-7 Verots S3 SP2/0 respectively showed the presence of recombinant GH protein in cultured supernatant of transfected cells (Min et al. 2009). The positive results of our expression studies in animal cell line model have provided the bases to proceed for determining the biological activity of rcGH-pND in animal model. Thus the results of weight gain and tibia width assays showed that rcGH-pND was biologically active in the muscle of animal and may be used as supplement to increase the biomass and meat production in goat breeds as well as in sheep breeds since
Table 1: Weight gain data of BALB/c mice from 1-4 weeks post inoculation of recombinant caprine growth hormone
(rcGH) in mammalian pND vector
Treatment###0 week###1st week###2nd week###3rd week###4th week
Table 2: Tibia width data of BALB/c mice from 1-4 weeks post inoculation of recombinant caprine growth hormone
(rcGH) in mammalian pND vector
Treatment###0 week###1st week###2nd week###3rd week###4th week
caprine growth hormone is identical to ovine (Ascacio- Martinez and Barrera-SaldaAa 2012). However effect of plasmid based rcGH for enhancement of milk production needs to be determined. The weight gain and tibia width assays has been used in a number of studies especially to evaluate the effect of human growth hormone in dwarf little mice (Bellini and Bartolini 1993) recombinant human growth hormone (George et al. 2007; Kwak et al. 2009) recombinant DNA derived human growth hormone/somatotropin in hypophysectomized rats (Zhu et al. 1997). The reason being that recombinant human growth hormone is used to treat growth hormone deficiency in children and adults and wasting in AIDS patients (Cox et al. 2007).Draghia-Akli et al. (2003; 2006) also used plasmidbased technology to deliver growth hormone releasing hormone (GHRH) a releasing hormone for growth hormone to various animal species for screening toxicology and therapy. A GHRH-expressing plasmid was engineered for efficient expression in skeletal muscle and following intramuscular injection enhanced by electroporation in piglets it was observed that GHRH is synthesized in injected muscle secreted into blood stream and stimulated normal pituitary GH production and release. The release GH then in turn stimulated the growth of piglets. In another study carried out by Meng et al. (2004) observed enhanced growth in sheep when plasmid expression vector having sheep GHRH was injected in muscle tissue of the animals. These studies indirectly support present study in which plasmid DNA of growth hormone rather than plasmid DNA of GHRH has been used to evaluate the biological activity. Thus all these studies (indirect) including ours (direct and first of its kind) support the notion that plasmid based gene delivery system is more feasible in terms of administrating plasmid rather than administrating the product of gene in purified form which is not cost effective. However further studies are required to determine the effect of plasmid-based delivery of GH rather than protein based delivery on milk production by farming animals.In conclusion the recombinant plasmid based technology seems to be a good source of gene delivery and in vivo transgene expressions. Further it is cost effective therapy in comparison to exogenous administration of recombinant homologous GH especially in resource limited setting to enhance the genetic potential of animals toincrease meat and milk production.
The authors are thankful to Dr. Garry Rhodes University of California Davis CA USA for providing mammalian expression vector pND. Further we are highly grateful to the Higher Education Commission Pakistan for generous financial assistance.
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|Publication:||International Journal of Agriculture and Biology|
|Date:||Jun 30, 2014|
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