The overall goal of this set of protocols is to detect alternative first exons in the mRNA of a gene such as Bcrp1 and to establish luciferase-based reporter assays for the putative promoters upstream of the alternative identified first exons. The in-silico methods used can help answer key questions in the transcription and regulation field such as how mRNA expression is controlled in different tissues. The main advantage of this technique is that it uses publicly available genetic data to identify alterative first exons in the mRNA of gene of interest, which implies alternative promoter usage.
This method that prepares report constructs and conducts reporter assays can help answer key questions in the gene regulatory field such as the functional activity of a putative alternative promoters. To begin this procedure, obtain the sequence for mouse Bcrp1 exon 2 by inputting the mRNA reference sequence ID into the search window of the Ensembl genome browser and click Go.Next, select a full length sequence that contains 16 exons. Click the Download sequence option.
Then, choose the FASTA format and click Preview. In the next screen, copy the preview sequence of exon 2 into the clipboard. Navigate to the blast homepage on the NCBI website.
Select Mouse genome. Paste the exon 2 sequence from the clipboard into the query box. After that, select ESTs from the Database drop down.
Optimize it for Highly similar sequences and then choose BLAST. When the BLAST run is complete, the results page will appear. Under the Descriptions subheading of the results page, select All, then Download, followed by FASTA in the drop down, and then choose Continue.
A txt file will appear. Open and copy the entire file into the clipboard. To identify the location of the EST sequence that is five prime to exon 2 in the Bcrp1 gene, on the BLAST homepage under the Specialized BLAST subheading, select Align two sequences using BLAST.
Paste the text file from the clipboard into the query box and enter 372099104 in the Subject Sequence box. Optimize it for Highly similar sequences under Program Selection and run BLAST. Once the results window appears, view the alignments graphically by clicking Graphics in the Alignments window.
Use the right and left arrows and zoom to focus on Bcrp1/ABCG2 and the sequence alignments. Then, save the sequence alignments by selecting All in the Descriptions box. Under the Download drop down, select Hit Table and then click Continue.
As the position of the five prime end of exon 2 corresponds to the nucleotide 58, 655, 638 in the chromosome six contig, designate this as plus one and then calculate the position of the partial sequences of each EST five prime to 58, 655, 638. A critical step in this procedure is the prediction of multiple first exons. The alternative promoters are hence identified here.
Using the sequence of E1U obtained from the EST database, designate the first five prime nucleotide of E1U as plus one. Obtain a back clone of mouse chromosome six that contains the Bcrp1/ABCG2 gene sequence and the sequence at least 100 kilobases upstream of the Bcrp1/ABCG2 gene. Then, identify a suitable reporter vector, such as the luciferase reporter plasmid pGL3-Basic.
Determine the multiple cloning sites in the reporter vector. KpnI, SacI, MluI, NheI, SmaI, XhoI, BglII, and HindIII are the restriction endonucleae sites in the multiple cloning site of the pGL3-Basic vector in the five prime to three prime orientation. Next, select the two kilobase region upstream of the E1U exon and paste the sequence in the DNA5 followed by running the program.
The details of the single and multiple digestion sites will be displayed. After that, compare the list of digestion sites on the E1U region with the MCS on the pGL3-Basic vector in an excel sheet. Subsequently, prepare forward and reverse primers for PCR that contain restriction endonucleae sites using commercial gene primer synthesis services or primer selection software.
Select a forward primer located about two kilobases upstream of the E1U sequence and a reverse primer within the E1U sequence. Then, amplify the E1U genomic region using these primers with PCR 01 to one microgram of Bak and PCR Master Mix containing high fidelity taq polymerase. Afterward, verify the length of the amplified PCR product by comparing it against a one kilobase DNA ladder using 0.8%TAE agarose gel electrophoresis.
Afterward, digest the obtained PCR product and the pGL3-Basic vector using restriction endonucleases specific for restriction digestion sites introduced into the forward and reverse primers. Then, verify the digestion of the PCR product using agarose gel electrophoresis. Cut the digested vector DNA and the two kilobase PCR band from the agarose gel.
Next, extract the digested vector DNA and the two kilobase PCR band from the gel slices using a commercial SV gel and PCR clean-up systems kit. Measure the concentration of the purified PCR product and the purified vector using UV spectrometry. Subsequently, prepare the E1U reporter construct by ligating the purified restriction enzyme digested PCR product and the pGL3-Basic firefly luciferase reporter vector using the T4 DNA ligase quick ligation kit thereby producing the E1U reporter construct named pGL3 E1U.
In this step, culture the TM4 cells in 24-well plates at an initial density of 200, 000 cells per well. Six hours after placing the cells in culture, transfect the cells with 0.2 micrograms of MP pGL3-Basic vector or 0.2 micrograms of pGL3 vector along with four nanograms of pRL-TK as an internal control. Thirty hours following transfection, remove the growth media from the transfected cells.
Wash the cells with PBS, then remove the wash solution. After that, measure the firefly and Renilla luciferase activity using a commercial kit. Express the activity for each tube as the ratio of the firefly luciferase activity divided by the internal control.
This step confirms the functional activity of the testis specific promoter. Here is the reporter assay for Bcrp1 promoters E1U, E1A, E1B, and E1C in bulbs these are totally cell-derived TM4 cells. Data are expressed as the luminescence of firefly luciferase normalized to that of Renilla luciferase.
The asterisk indicates a significant difference from the MP pGL3 vector control. This graph shows the effects of mutation of the SF-1 response element in the Bcrp1 E1U reporter construct on luciferase activity. Bcrp1 reporter constructs with a putative SF-1 binding region were transfected into TM4 cells with and without the enforced expression of SF-1.
In the figure, a denotes a significant difference from the empty vector pGL3 control;b signifies a statistically significant difference compared to the E1U promoter construct in the vector transfected cells. While attempting this procedure, it's important to remember to select an appropriate cell line for the reporter assay. The cell line should express the gene of interest under the control of the promoter in the reporter construct.
Following this procedure, other methods, such as quantitative RT-PCR can be performed in order to facilitate evaluation of alternative promoter usage in multiple tissue samples. After watching this video, you should have a good understanding of how to predict alternative promoter usage for a gene of interest and to determine the functionality of that promoter.
