Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2008 Analysis of the Porcine TAP1 promoter: insertion into pCR2.1 plasmid and looking for sequence variants. Program supported by the National Science Foundation Research Experience for Undergraduates DBI Whitney Davis ab, Oliver Couture b, Jolita Uthe b, Chris Tuggle b A Department of Biology, Alcorn State University, Alcorn State, MS, 39096, USA b Department of Animal Science, Iowa State University, Ames, IA, 50010, USA Figure 4: By using double the volume of culture yielded twice the amount of DNA TATA Box Transcription Factor Binding sites Gene 1 Gene 2 NFkB Exons Intron Figure 1: Gene and Promoter Promoter TOPO cloning Vector PCR2.1 Plasmid Figure 2: Insertion Process Salmonella is an infection in animals that can lead to death in animals and very serious illness in people Farmers lose millions of dollars a year due to salmonella infection Can lead to food borne diseases and salmoneloisis. We are testing promoters to see if NF-kB is important for the expression of TAP1 We are using pCR2.1 to emulate pigs and their immune system to check if NF-kB is important in the expression of TAP1. This can will soon help reduced the amount of pigs infected with salmonella by increasing and selecting for the correct immune response in pigs. Using Marker Assisted Selection to find difference s in shedders and non-shedders in the TAP 1 gene PART 1: TOPO kit: Concentrations after kit: pCR2.1 sample ng/mL, sample ng/mL, sample ng/mL, and sample ng/uL. A second run was used for more product and the results were as follows, sample ng/mL, sample ng/mL, sample ng/mL, and sample ng/mL. See Figure 4. Checking Plasmid vectors: pCR2.1 is cut at 508 & 2550, and pGL3 cuts at 3523 & 4569 (Figure 5) Checking insert in pCR2.1: Came out with bands in PCR, but needs to be repeated Checking Orientation of insert: Needs to be repeated PART 2: PCR and Purification of shedders/non-shedders, and pools All PCR reactions worked or were re-done with the predicted 200 base pairs insert All of the samples had enough DNA to be sequenced for the experiment Sequenced Product SNP was detected at the 425 consensus position C= 85% and T= 15% Three different allele types show in non-pooled pigs C/C, C/T, and T/T Pools also showed variation: pool 1-C/T, pool 2-C/T, pool 3-C/C, and pool 4-C/T Sequences came out to be around 450 base pairs PART 1: Pig 133 was chosen at random for the DNA insert that was needed. The PCR that was preformed was very successful in getting the TAP1 insert out the pig genomic DNA The TOPO kit proved a easy way to insert the promoter into the pCR2.1 vector Since both pCR2.1 and pGL3 were cut at the correct position our insert and our restriction enzyme worked correctly The other two tests were faint proof and need to be redone to further prove insert. All of tests did not confirm insert; more study will have to be done PART 2 PCR confirmed that primer s work very well with genomic pig DNA There was a SNP in the sequences showing variation between pigs Three different allele types were present C/C, C/T, and T/T Only variation was among shedding animals 115 and 142. More study will have to be done to show characterization of the SNP PART 1: Primer Design Design of primers to amplify the TAP1/PSMB9 bidirectional promoter were previously done by Tuggle lab PCR of 40 pigs The DNA of 40 pigs was isolated by Tuggle lab PCR was performed to make sure primers worked correctly, pig number 133 was chosen out of the pigs for cloning. PCR for TOPO cloning Procedure was taken out of TOPO Cloning kit The PCR included 85ng of DNA template from animal 133 from Tuggle lab Promoter into TOPO cloning vector pCR2.1 The PCR insert from pig 133 was used with the TOPO Cloning kit to insert the promoter fragment into the vector. Checking insert Cut with PvuI pCR2.1 is cut at 508 & 2550 and pGL3 cuts at 3523 & 4569 A regular digestion of two hours was carried out PCR of PCR2.1 We used samples 1-4 of the direct plasmid and bacteria we recovered out of the TOPO kit. Check orientation with PCR We used sample 1-4, This was done by using the following primer combination: pig primer forward & pig primer reverse, pig primer forward & sequencing primer forward, pig primer forward & sequencing primer reverse, pig primer reverse & sequencing primer forward, and pig primer reverse & sequencing primer reverse in PCR. PART 2: PCR of 4 non-shedders, 6-shedders, and four pools of pigs A 25uL reaction was used with all 27 pigs DNA Purify Quantified PCR product Used Qiagen PCR purification kit Sequenced product Samples were diluted to the specification of the Iowa State DNA sequencing center. SNP in TAP1 promoter Sequence 115: GAGT T TGGG Sequence 134: GAGT C TGGG Sequence 142: GAGT C/T GGG Figure 3: pCR2.1 Vector map Wang Y., Qu, L., Uthe, J.J., Bearson, S.M.D., Khuar, D., Lunney, J.K., Couture, O.P., Nettleton, D., Dekkers, J.C.M., and Tuggle, C.K Global transcriptional response of porcine mesenteric lymph nodes to Salmonella enterica serovar Typhimurium, Genomics. doi: /j.ygeno N. Hadjeb and G.A. Berkowitz, Preparation of T-over-hang vectors with high PCR product cloning efficiency, BioTechniques 20 (1996), pp. 20–22 Chen Q., Zhou H., Chen J., Wang X Using a modified TA Cloning method to create entry clones. Analytical Biochemistry. Volume 358, Issue Wei L., Sandbulte M., Thomas P., Webby R., Homayouni R., Pfeffer L., 2005, NFkB negatively regulates interferon-induced gene expression and anti-influenza activity. J. Biol. Chem., /jbc.M Figure 5: PvuI confirms the correct plasmid sizes pGL3 p CR2. 1 Pig 115: T/T Pig 134: C/C Pig 142: C/T SNP Figure 6: SNP in sequenced DNA