The Cloning and Sequencing of the Regulatory Element of Goat Beta-Casein Gene

So far, many promoters have been isolated and used to drive the expression of foreign gene in the mammary gland. However, none of these promoters were specific and the expression in the mammary gland tissue was associated with expression in other tissues. The need for discriminating sequences, which can drive the expression of foreign gene specifically in the mammary gland, is obvious. This study is intended to clone specific regulatory elements of 11 casein gene, which may drive the expression of every gene specifically in the mammary gland. The isolation and purification procedure of the genome goat from the mammary gland and the isolation and purification of Lambda vector was performed. The right and left arms were of the phage were also purified and packaged with packagene. A goat genome library consisting of 2. 1 X 10 phages was constructed and screened by using in situ hybridization technique. Following the procedure of Sambrook et al. (1989) we performed the in-situ hybridization technique. The probe was prepared through Polymerase chain reaction (PCR) using the goat genome as template. The probe was used in the in-situ hybridization technique to screen the library after cloning it into puc 18 vector. Sall and BamHI were used for the analysis of the recombinant phage. The sal 1 fragments were cloned in pGEM5z vector. The partial sequencing of the 4.5kb long Sal 1 fragment cloned in pGEMbz was performed following the procedure of Sambrook et al. (1989). In this study we suggest that the goat 11 casein promoter may be very strong and tissue specific promoter, which may contribute in the progress and development of the mammary gland expression for the production of valuable proteins in the milk. Introduction The easy automation of DNA sequencing has greatly facilitated in the characterization of mammary cDNA and genes associated with milk proteins in various species. Some 60 cDNA and 20 genes from 12 species have already been completely sequenced (Bonsing et al., 1988). These cDNA or genes can now be modified in vitro by site directed mutagenesis and then expressed in various systems, such as bacteria, yeast, Bculovirus infected cells and Cos cells after insertion into adequate vectors. Studies of genetic polymorphism at the nucleotide level have led to the discovery of new alleles (Roberts et al., 1992). Expression analysis of native or modified genes using in vitro transcription systems, mammary cell lines, and transgenic animals has greatly improved the knowledge of the functioning of milk protein and has offered the potential for the genetic modification of milk composition (Persuy, 1992). Over the last years, using biotechnology, researchers developed mice, which demonstrated promoter elements with tissue specificity driving on hormonally regulated fusion DNA to express products, (Reddy, 1991) however, only a limited number of promoter elements have been introduced in to domestic farm animals. Materials and Methods A high molecular weight DNA was isolated from lactating goat mammary gland by using methods described by Sambrook et al., 1989. It was not an easy task to get very pure goat genome DNA and the required length of the fragment. By using phenol and ether, highly pure Genome was extracted, which was between 23 and 19kb long. First Enzymatic Digestion Pilot Experiment was performed to establish the exact ratio of enzymes to DNA required to fully digest the DNA then Sau3 was used to digest the DNA. In order to isolate the required length of fragments from the rest, the Sucrose Gradient Centrifugation technique was applied (Sambrook et al., 1989) and analyzed by electrophoresis by using 8 HindIll Marker as indicative of size. All the tubes which contained fragments 23<19kb long were combined and used for further construction of the Genome library. Before constructing the library it was also necessary to purify the fragments because recovered fragments by Sucrose Gradient centrifugation contained sucrose. Elutiploid and 2X low salt and high salt buffer were used to purify the fragments. After the elution was completed the DNA fragments were precipitated with 2 volume of ethanol and recovered by centrifugation at 12000 g for 10 minutes and dissolved in 50 μl TE pH 8.00 and were stored in -20EC. At this stage purified fragments were ready to be legated into a vector 8 arm. The bacteriophage EMBL3 was the phage used to prepare pure 8 DNA. The EMBL3 phage was selected because it can accommodate large fragment of genome DNA, up to 20 Kb, and because this vector is particularly useful for cloning sau3 partial digests (Sambrook et al., 1989) and also because BamHl sites in the vector are flanked by Eco RI and Sall sites and, therefore, it was possible to release the cloned fragment by digesting the recombinants with Sail and EcoRl. It was not ideal to clone the fragment into the plasmid vector as the fragment was 23 Kb <19Kb, therefore, the plasmid vectors could not accommodate it. To get a vector, which can accommodate such large fragments, isolation and purification of 8 DNA from EMBL3 phage was performed following the method of Sambrook et al. (1989) and digested with Bam HI and the results of the