preferably , the muscle specific gh expression system is constructed in a plasmid vector . the complementary dna ( cdna ) sequences encoding ghrf signal peptide and ghrf itself were designed from the published peptide primary structure and optimized for mammalian codon usage . this cdna is synthesized using conventional oligonucleotide synthesis chemistry . the ghrf and its signal peptide encoded by the cdna sequences can be the corresponding natural or recombinant or synthetic , or biologically active fragments or their analogues with similar activities . the ghrf leader sequence encodes a signal peptide in the nascent ghrf peptides that will direct the export of said ghrf peptides out of the muscle cells and secretion into the general circulation in an active form . the dna sequences for promoter / enhancer elements and the three prime ( 3 ′) untranslated , polyadenylation signal containing regions of the promoter / ghrf gene is separately cloned by polymerase chain reaction . as an example , the skeletal α - actin ( skαa ) promoter / enhancer is used but this should not be considered as a restriction to the choice of promoter / enhancer . the full set of , but not a minimal subset of such an actin promoter as described by draghia - akli et al , is used . the reason is that in vivo studies using direct gene injection have shown that the full skeletal α - actin promoter gave the highest level of gene expression in the mouse muscle when compared to other truncated versions fleecy et al ., animal biotechnology 2 : 101 - 120 , 1998 ). as a preferred embodiment of this invention , the dna sequences of the actin promoter , the cdna sequence encoding a ghrf signal peptide , and the cdna sequence encoding gbrf are species specific . these segments of dna are inserted into a puc plaid in the following order : actin promoter ; human ghrf signal peptide cdna ; porcine ghrf ( 1 - 44 ) cdna ; actin 3 ′ untranslated region . the puc plasmid also contains a gene for antibiotic resistance , for instance ampicillin resistance , that allows selection when transformed into e . coli bacteria , as well as a colei origin of replication that confers high plasmid copy number . [ 0034 ] e . coli transformed with the ghrf expression plasmid is then grown in a fermenter under ampicillin selection . after harvesting the cells and release of plasmids by alkaline lysis , the plasmid dna is separated from the pna , the bacterial dna and other cellular contaminants by column chromatography . the integrity of the purified plasmid dna is verified by restriction enzyme digestion / agarose gel electrophoresis and dna sequencing . purity is determined by measuring absorbance at od 600 / 280 and agarose gel electrophoresis . assayed plasmid dna is dissolved in a physiologically acceptable carrier , tested for pyrogen levels , dose adjusted and administered to animals via intra - muscular injection . as a preferred embodiment of this invention , the total amount of said plasmid dna given per animal should be between the range of 1 to 100 μg per kg body weight of the livestock . upon injection into the muscle of the animals , the plasmid expression vector , being a dna , is taken up into the muscle cells . the plasmid expression vector will then direct the cell to produce ghrf peptides . such peptides are released into the circulation system and stimulates the production of gh on reaching the pituitary gland . this results in the elevation of oh in the circulation system that improves growth performance , results in faster growth rate , higher lean - body mass and better food conversions . furthermore , the meat produced is leaner and with less fat , hence of a higher market value . furthermore , the normal negative feedback control of gh via the hormone somatostatin is bypassed , as ghrf release is under the control of a constitutive muscle specific promoter . this allows the continuous production of gh . the following examples are offered to further illustrate but not limit the embodiments of the present invention . the pghrf expression construct was designed to give optimal production and secretion of the pghrf peptide when transfected into mammalian muscle cells ( fig1 ). skeletal alpha - actin ( skαa ) promoter is a well characterized promoter that has been shown to give highly specific gene expression in differentiated muscle fiber cells ( reecy et al , gene 180 : 23 - 26 1996 ). furthermore , it possesses a high degree of sequence identity and share many transcription factor consensus binding site comparing with the human , bovine and chicken skαa promoters . this suggests that this promoter is highly conserved between mammals and birds . this is supported by in vitro studies demonstrating the ability of the porcine skαa promoter to confer muscle specific gene expression in mouse , rat and porcine cells . hence this 2 kb promoter was chosen to drive the muscle specific expression of the pghrf cdna . the porcine skαa promoter was spliced in front of an open reading frame that comprises the dna sequence coding for the human ghrf signal peptide and the porcine ghrf mature peptide . since the dna sequence encoding porcine ghrf was not available , we designed this by reverse translation of the known peptide structure , with optimization for mammalian codon preference . a further modification of the present invention involves the addition of the 3 ′ untranslated region of the porcine skeletal α - actin gene downstream of the coding region . this serves to enhance expression of the pghrf product by stabilizing and prolonging the half - life of the mrna transcripts . the method of construction for the pghrf expressing plasmid is detailed below and zed in fig2 . polya region : genomic dna was purified from porcine kidney and used for amplification of porcine gene sequences . a 750 bp 3 ′ end fragment of the skαa gene was amplified by pcr . this region spans nucleotide number 2464 to 3204 of the published sequence ( genbank accession no . u16368 ) and contains the 3 ′ untranslated region and putative polyadenylation signal . the product was then cut with restriction enzymes xba i and sac ii ( using cuts sites introduced with the pcr primers ) and cloned into plasmid vector pbluescript ks + to get psαapa . ghrf coding region : the ghrf coding region is an open reading frame ( orf ) that comprises the dna “ leader sequence ” encoding a signals peptide for human ghrf succeeded by an inframe porcine ghrf ( 1 - 44 ) cdna . the leader sequence , containing full exon 2 and partial exon 3 of the hub an ghrf gene ( genbank accession no . l100345 ) was pcr amplified from human genomic dna . the porcine ghrf cdna was constructed as two oligonucleotides with the following sequences : these oligonucleotides contained an overlap that was used to anneal them to each other and then filled in with vent polymerase . the annealed product was then pcr amplified with vent polymerase . the products of the leader sequence and porcine ghrf cdna were each kinased with t4 polynucleotide kinase and then purified by polyacrylamide gel electrophoresis . then the products were joined together with t4 dna ligase and the ligation product of 265 bp further purified using polyacrylamide gel . this orf product was then re - amplified by pcr with primers containing xma i and xba i restriction enzyme sites . the orf was then cut with xma i and xba i enzymes and purified to get a 245 bp fragment . this fragment was then cloned into psαapa to get ppgrfpa . promoter region : the full 2 kb porcine skeletal α - actin ( skαa ) promoter / enhancer was pcr amplified from porcine genomic dna . nested - polymerase chain reaction ( pcr ) was then done on { fraction ( 1 / 50 )} of the first pcr reaction to yield a dna product that spans bp − 1909 to + 76 of the porcine skαa gene ( reecy et al , gene 180 : 23 - 28 , 1996 ). this product was blunt - ended with t4 dna polymerase and then cut with eco rv . the gel - purified promoter fragment was cloned into the eco ri and eco rv sites of ppgrfpa to get the intermediate construct psαapgrfpa . to obtain the final construct , this expression cassette was then excised with flanking kpn i and sac i restriction enzymes and ligated to plasmid puc19 , and designated pupagrf . a second construct was made by removing the entire pghrf coding region from plasmid pupagrf by partial digest with xho i ( fig3 ). this second plasmid , pupax , was used as a control in the animal trials . the plasmids were separately transformed into e . coli k - 12 ( strain dh5α ) and grown in a fermenter using culture media supplemented with 150 μg / ml ampicillin . when the culture reached stationary phase , the bacterial cells were harvested and lysed by classical alkaline lysis . the plasmids were recovered by centrifugation and separated from contaminating cellular dna , rna and proteins by anion exchange chromatography . the purity was assayed by measuring the absorbance at 260 and 280 nm . the purified plasmid dna was also checked for integrity by agarose gel electrophoresis . finally , identity was confirmed by restriction enzyme digestion and dna sequencing . the plasmid dna was precipitated and redissolved in pyrogen free phosphate buffered saline ( pbs ) and concentration calculated by taking the absorbance at 260 nm . after passing pyrogen tests , the dna was ready for animal injection at this point . to test the ability of the pupagrf construct to enhance growth performance , we injected this plasmid in the quadriceps muscle of the mouse . from a breeding colony of c57bl / 6j mice , forty - eight male mice of 14 . 5 - 18 g body weight were selected and randomly assigned to four groups of twelve . each group of 12 mice were given a single injection of 100 μl pbs containing either 100 μg of control plasmid , or ghrf expression plasmid ( pupagrf ) at 30 , 100 or 200 μg , respectively . the mice were weighed , toe - marked for identification , then given a single injection in the middle of the left quadriceps muscle . the mice were kept on a 12 hour daylight cycle under a controlled climate , minimal disease environment and given standard rat chow and water . body weights were recorded twice per week . the mice were anesthetized with ketamine / xylazine ( 0 . 15 ml per 100 g body weight ) to obtain blood and tissue samples . blood was collected by cardiac puncture into a 1 ml insulin syringe containing 30 μl of 0 . 5 m edta as anti - coagulant . collected blood was kept in ice until plasma was obtained by pelleting the cells at 6000 g for 10 min . the entire quadriceps muscle , which included the injection site was dissected out and snap frozen in liquid nitrogen . the right lobe of the liver was also dissected and immediately frozen . all samples were stored at − 80 ° c . until further analyzed . mice in all four treatment groups showed rapid growth and increase in body weight in the four week period after injection . in the 21 day period post injection ( p . i ), body weight values were not significantly different between all groups . although mice given 30 μg pupagrf showed the highest gain in weight whilst the highest dose ( 200μg ) group lagged behind ( fig4 ), by 25 days p . i ., there were significant differences between the higher doses and the control . at 25 days p . i ., mean body weights for the 200 , 100 and 30 μg groups were 26 . 3 , 26 . 5 and 24 . 7 g respectively , whilst that for the control group was 24 . 5 g ( p & lt ; 0 . 032 , single factor anova ). mean body weights at 28 days p . i . were 26 . 8 and 26 . 6 g for the 200 and 100 μg treated mice , versus 24 . 5 and 25 . 0 g for the 30 μg and control treated , respectively . again , there is significant difference to the weights of the control treated mice ( p & lt ; 0 , 0045 ). the effects of the pupagrf injections on the percentage weight gain are shown in fig5 . four weeks after plasmid injections , control treated mice showed percentage gain of 48 . 7 %, those at 30 and 100 μg of pupagrf of 56 . 6 % while those at 200 μg of 68 . 4 %. these results indicate that a single injection of a pghrf producing plasmid is able to produce a significant improvement in the growth rate of young mice . at doses up to 100 μg , there was an improvement of approximately 8 % whilst a dose of 200 μg was able to give a 19 % improvement in the percentage weight gain . these results are comparable to the observations of draghia - akli in the regenerating mouse muscle , while their injection methods required a prior injection of the myotoxic bupivacaine to enhance plasmid uptake and expression . we then tested the ability of the pghrf gene treatment to improve growth performance in farmed pigs . breeder pigs of a triple cross from landrace / yorkshire / duroc ( yd ) were used in this study . young male pigs were randomly assigned into three adjacent pens on a breeder farm at 10 weeks of age . pigs were given the plasmid preparation by a single injection in the gluteus muscle with a 1½ inch 16 gauge syringe . pigs in pen a and b were injected with 1 mg and 4 mg of pupagrf respectively , while those in pen c were given 4 mg of the control plasmid pupax . the pigs were given feed and water ad libitum and weighed weekly . pigs given a single injection of 1 mg of pupagrf showed a weight gain from 62 . 53 to 87 . 17 kg ( increase of 37 . 8 % o ) in the 6 weeks period of the experiment . pigs injected with 4 mg grown from mean weight of 60 . 20 kg to 98 . 28 kg ( gain of 44 . 1 %). those in the control group grown from 58 . 59 to 89 . 33 kg ( a 34 . 4 % gain ) in the same period . thus after 6 weeks , those pigs injected with the pghrf expression plasmid showed a higher percentage weight gain than those injected with the control plasmid . furthermore , there seems to be a trend for a dose dependent response since the 4 mg dose caused over two - fold improvement compared to the 1 mg dose . in terms of feed conversion efficiency , the 1 mg dose group , although showing a modest gain in growth rate , consumed 10 % less feed ( table 1 ). moreover , there was no difference in the amount of feed consumed between the control and high dose group , hence pupagrf at both 1 mg and 4 mg doses were able to give an improvement in feed conversion . studies were conducted to determine the effects of injecting 2 mg of pupagrf plasmid to landrace / yorkshire ( ly ) pigs on the accumulation of back - fat . twenty ly pigs at approximately 32 weeks old and average weight of 55 kg were assigned to two groups of ten , each with 5 males and 5 females . one group was administered 2 mg of pupagrf plasmid by intramuscular injection . the second group was similarly treated with the plasmid , pupax , and served as a control . back - fat thickness at positions lateral to the last thoracic vertebra was determined weekly using a renco ultrasound meter . three measurements were taken at positions pi , pii , and piii being 45 mm , 65 mm , and 80 mm lateral to the center of the spine at the last rib vertebra respectively , and averaged to give a mean back fat thickness . the results are summarized in fig7 . in mates , both pghrf treated and control pigs showed gradual increases in back fat thickness as the animals aged , although those , given the pghrf expressing plasmid showed a reduced gain in back - fat . female control pigs also showed a gradual gain in backfat , which is suppressed by pghrf treatment . table 2 shows the relative gains in back fat thickness over the experimental period . at 60 days post injection , pghrf treatment reduced the amount of back - fat accumulation in both male and female ly pigs . the results of the examples show that treatment with the pghrf gene is able to produce benefits in growth performance in both the mouse and pig . the gains in growth rates are comparable to those of gh treatments yet with a much more cost effective treatment regime . the quantity of dna used per animal is at least 100 fold lower than that of growth hormone injection reported in the literature . furthermore , the pig data shows that there are reductions in the amount of feed required as well as the thickness of back - fat . therefore this invention will be beneficial to the livestock industry by enhancing feed efficiency and growth rate of livestock . while the preferred embodiment of the present invention has bee described in detail by the examples , it is apparent tat modifications and adaptations of the present invention will occur to those skilled in the art . it is to be expressly understood , however , that such modifications and adaptations are within the scope of the present invention , as set forth in the following clams . furthermore , the embodiments of the present invention shall not be interpreted to be restricted by the examples only .
this invention relates to the enhancement of feed efficiency and growth rate , and the reduction of fit accumulation to produce better quality meat by administration of an exogenous gene sequence comprising the complementary dna sequence of growth hormone releasing factor to stimulate the production of the endogenous hormone peptide growth hormone . this invention further relates to methods for producing such gene sequence .