A0130765h_lab Report 1.pdf 6o526r

LSM2191: LABORATORY TECHNIQUES IN LIFE SCIENCES SEMESTER I, AY2015/2016

Molecular Cloning of LDHA Gene from Mus musculus Fibroblast Cells BY SEAH CHUN LING JOVENA A0130765 LS LAB 8, GROUP 27 TEACHING ASSISTANT: HILARY HO KUNG YU

Abstract (200 words)

Lactate Dehydrogenase A (LDHA) is a subunit of lactate dehydrogenase (LDH) and plays an important role in the development of cancer cells. LDHA catalyses the conversion of pyruvate to lactate in glycolysis and cancer cells utilise this pathway to meet its energy requirements for cell proliferation and hence tumour growth. Due to this ability of cancer cells to proliferate with LDHA, studies on the oncogenic nature of LDHA has been researched extensively to develop a potential cancer therapy targeting LDHA expression levels. In this experiment, mouse fibroblast cells were used in the extraction of LDHA mRNA, which was then reverse transcribed and amplified. The gene was then inserted into pET11a plasmid vectors. The plasmids were then introduced into DHSα competent E.coli cells for gene amplification. Recombinant cells were identified and the plasmid DNA was extracted and sent for DNA sequencing. The sequencing results had a 99% match to the reference sequence in the database. Further analysis of the LDHA gene can now be done.

1 Introduction

The leading cause of death in Singapore for the past three years has been cancer, averaging 30% of all deaths within the country (Ministry of Health, 2015). The fourth most common cause for cancerrelated death in the world is pancreatic ductal adenocarcinoma (PDAC) and while diagnosis and treatment research has been done, survival rates have not been improving (Rong et.al, 2013). Researchers are thus accelerating research to determine the root cause and come up with new treatments. LDHA has been shown to play a role in cancer cell development as in various forms of human cancer, expression of LDHA was found to be upregulated (Shim et.al, 1997). LDHA promotes pyruvate to lactate conversion, providing energy in the form of adenosine triphosphate (ATP) during periods when oxygen is scarce and anaerobic metabolism takes over. LDHA is important in sustaining the glycolytic phenotype of cancer cells. When expression of LDHA is knocked down, inhibition of cell growth and cell migration occurs, reducing formation of tumours (Yao et. Al 2012). Studying LDHA is thus an attractive strategy in combatting cancer. Furthermore, ongoing studies have been promising as researchers are conducting tests to determine if the knocking down of LDHA expression can lead to the apoptosis of pancreatic cancer cells (Rong et.al, 2013). As such, an in depth understanding of the LDHA gene on a genetic and molecular level is required and this paper aims to achieve it. In this experiment, 2x106 Mus muculus fibroblast cells were used to sequence the LDHA gene. Mouse cells were used because approximately 95% of their genomes are similar to humans. Genetic research on mice can be thus applied to human diseases (Jackson Laboratory, 2014). To sequence the gene, we have to undergo certain steps. To extract total RNA, TRIzol® extraction technique was used. Phenol and chloroform present in the reagent separates the cell contents after centrifugation into an aqueous and organic phase, the former of which total RNA was extracted from. However, precautions have to be taken to avoid contamination as RNase present in the environment can degrade RNA. Autoclaved equipment and solutions treated with chemical diethylpyrovarbonate (DEPC) were used, gloves were worn at all times and RNAqualified RNase-free DNase was also used to ensure that the integrity of the RNA is maintained in the process of removing DNA. After RNA extraction, Reverse Transcription – Polymerase Chain Reaction (RT-PCR) was conducted. cDNAs were synthesised from the mRNA of the isolated total RNA. PCR allows for the rapid amplification of specific DNA fragments from a trace amount of DNA in vitro. It is also highly specific and the primers determine which region should be amplified. Taq DNA polymerase was used as its heat stability allows repeated cycles of denaturing at 95°C. DNA ligation and transformation was then carried out to construct recombinant plasmids. Both plasmids and DNA inserts were cut with the same restriction enzymes (RE) to ensure complementary ends. To prevent re-ligation, plasmids were cut with another RE to produce blunt ends. DNA ligase was added to ligate the plasmid and inserts together and the recombinant plasmids were introduced

into competent cells treated with CaCl2 that increases cell membrane permeability. Resultant cells are then grown on agar plates. Extraction of the plasmid DNA of the selected colonies were done using Wizard miniprep kit from Promega following the small-scale preparation protocol to determine if the plasmids were recombinant or not. Cell resuspension buffer includes RNase A, which prevents RNA contamination, and ETDA which removes cations, rendering nucleases inactive thus conserving DNA. Alkaline cell lysis buffer was added to break down the cell membrane and denature the DNA. Neutralisation buffer containing potassium acetate was then added to increase the pH of the solution, allowing plasmid DNA to renature while chromosomal DNA remains denatured, making it easy to wash off the chromosomal DNA. Wash buffer was then used to clean up the plasmid and nuclease-free water was used to elute the plasmid DNA. The plasmids then underwent restriction digestion and agarose gel electrophoresis to check for the presence of inserts. Plasmids with inserts were then sequenced using cycle sequencing to determine the exact nucleotide sequence of the insert. Results can then be compared to the database and analysed for mutations. Other than achieving our aim of sequencing the LDHA gene for further analysis, through these four practical sessions, we also familiarised ourselves with the steps and principles behind the steps of molecular cloning.

2 Material and Methods 2.1 Isolation of total RNA from mouse fibroblast cells TRIzol® extraction technique (Thermo Fisher Scientific) was used to extract the total RNA. The amount of isolated RNA was then quantitated using NanoDrop. 2.2 Reverse Transcription – Polymerase Chain Reaction (RT-PCR) 2.2.1 Reverse Transcription Oligo-dT primers anneal to mRNA, selecting mRNA from total RNA. After selection, reverse transcription was carried out using reverse transcriptase (RevertAid) to synthesise single stranded cDNAs. In total, four tubes were incubated with premixed enzyme cocktails, two RT (+) and two RT (-) for a total of 20μl, of which 2μl is RNA. 2.2.2 Polymerase Chain Reaction PCR was conducted using NDeI-H6-MmLDHA forward primer, EcoRI-MmLDHA reverse primer and Taq DNA polymerase for a total of 22.50μl under the following conditions – 95°C for 30 seconds (denaturation), 45°C for 60 seconds (annealing) and 72°C for 60 seconds (DNA synthesis). This was repeated for 25 cycles. 2.2.3 Agarose Gel Electrophoresis Agarose gel electrophoresis was carried out using 1% agarose gel with EtBr/SYBR safe in 1x TAE buffer at 100V for 45 minutes to visualise 5μl of products from RT-PCR. The resulting bands were observed under UV-light using the gel documentation system. 2.2.4 Clean-up of PCR fragment The RT-PCR products are purified by using binding buffer, washing buffer and centrifugation and then quantitated using NanoDrop. 2.2.5 Restriction Endonuclease Digestion The RT-PCR fragment (20μl) is mixed with two restriction enzymes (NdeI and EcoRI) and the pET11a plasmid (20μl) is mixed with three restriction enzymes (NdeI, EcoRI and BamHI). Both were incubated at 37°C overnight.

2.3 Construction of Recombinant DNA Molecules via DNA Ligation and Transformation 2.3.1 Clean-up of cut DNA fragments RT-PCR fragment and pET11a plasmid underwent double and triple restriction digestion respectively as stated previously. Resulting products were purified with same clean-up technique as Section 2.2.4 and quantitated using NanoDrop 2.3.2 DNA Ligation T4 DNA ligase were added to different volume ratios of pET11a: RT-PCR for ligation of RT-PCR products to pET11a plasmid vector and incubated at room temperature for 1 hour. 2.3.3 Transformation of Ligated Recombinant DNA Molecules into Bacterial Cells Ligated recombinant pET11a plasmids were transformed into DHSα competent E.coli cells (30μl) via heat shock method where the tubes were on ice for 30 minutes then heated at 42°C for 90 seconds. SOC medium was added to each tube then incubated at 37°C with shaking for 1 hour. The cells were then plated on individual LB plates containing 100μg/ml ampicillin and incubated at 37°C overnight. 2.3.4 Small-scale Preparation of Plasmid DNA From the prepared E.coli cells, recombinant plasmids were isolated by the small-scale preparation protocol using Wizard miniprep kit from Promega. Four plasmid samples were prepared and purified then quantitated using NanoDrop. 2.3.5 Digestion of plasmid DNA Purified DNA plasmids were double digested with EcoRI and NdeI with incubation at 37°C for analysis. 2.3.6 Agarose gel electrophoresis Agarose gel electrophoresis was carried out using 1% agarose gel with EtBr/SYBR safe in 1x TAE buffer at 100V for 45 minutes. 10μl of digested plasmids were loaded. The resulting bands were observed under UV and photographed. 2.4 DNA Sequencing 2.4.1 Preparing Cycle Sequencing Reactions and Purifying Extension Products Cycle sequencing reactions were set up using four different primers: pET11aF, LDHA 430-450, pET11aR and LDHA 570-550. PCR was then conducted under the following conditions – 96°C for 30 seconds, 50°C for 15 seconds and 60°C for 4 minutes for a total of 25 cycles. The PCR machine was set to hold at 4°C. The remaining procedures were done by professionals. 2.5 Sequence Analysis with BLAST BLAST was used to analyse and interpret our sequencing results which were done by First BASE Laboratories. Sequences were aligned to form a complete plasmid sequence and used to identify any discrepancies and determine if it is a sequencing error or a mutation.

3 Results 3.1 Quantitation Results: NanoDrop readings – RNA Isolation and RT-PCR Table 1: Quantitative results of isolated RNA and cDNA from RT-PCR RNA 1 RNA 2 A260/280 1.68 1.89 Concentration (ng/μl) 2854.3 925.5

cDNA 1.95 20.1

As shown in Table 1, the A260/280 reading for RNA 1 and 2 are 1.68 and 1.89 respectively, which is below the recommended value of 2.0 to classify as pure RNA. The concentration for RNA 1 is 2854.3ng/μl, which is higher than that of RNA 2 at 925.5ng/μl. Following RT-PCR, the cDNA obtained had a ratio of 1.95, which is considered pure as it is above the recommended ratio of 1.8, and a concentration of 20.1ng/μl. 3.2 Agarose Gel Visualisation – RT-PCR Products

Figure 1: Gel Image of Gel Electrophoresis of RT-PCR Products. Lanes 3 & 5 contains RT (+) enzyme cocktail while lanes 4 & 6 contains RT (-) enzyme cocktail. Lane 1 contains 1kb DNA ladder. As seen in Figure 1, lanes 3 & 5 showed one distinct band each at around 1000bp according to the DNA ladder while lanes 4 & 6 did not. This is true to our expectations as only lanes 3 & 5 had RNA mixed with the RT (+) enzyme cocktail. Lane 2 is empty hence no bands were observed. 3.3 Standard Curve for RT-PCR Table 2: Tabulated Values of Standard Curve for RT-PCR Band Size (bp) LogmW Distance from well (mm) Distance from gel end (mm) DNA 3000 3.477 33 70 Ladder 2000 3.301 37 70 1500 3.176 41 70 1000 3.000 47 70 750 2.875 51 70

Rf value 0.471429 0.528571 0.585714 0.671429 0.728571

Standard Curve of RT-PCR Products 3.6

3.477

3.5

LgmW

3.4

3.301

3.3

3.176

3.2 3.1

3

3 2.8

2.875

y = -2.2807x + 4.5277 R² = -0.9939

2.9 0.45

0.5

0.55

0.6

0.65

0.7

0.75

Rf

Figure 2: Standard Curve of RT-PCR Products. LgmW was plotted against Rf, with equation y = -2.2807x + 4.5277 and R2 value of 0.9939. A standard curve of LgmW against Rf for RT-PCR products was plotted using five different markers from the DNA ladder. LgmW is lg(size) and Rf is the distance from the well to the marker divided by the distance from the well to the end of the gel. Calculated values are shown in Table 2 and the standard curve was drawn as shown in Figure 2. As we can see from the curve as well as the R2 value of -0.9939, there is a strong negative linear correlation between the size and mobility of DNA fragments. The larger the size of the fragment, the slower the mobility and hence distance travelled. From Figure 1, we can see that in lanes 3 and 5, there is one distinct band each, roughly 46mm from the well, close to the 1000bp marker. Using the equation y = -2.2807x + 4.5277, where x = 47/70, we obtain a y-value of 2.996. The molecular weight of the band can then calculated by 102.996 = 991bp, which is close to our expectation of 1000bp. 3.4 Quantitation Results: NanoDrop readings – Restriction Digestion Products Table 3: Quantitative results of double digested RT-PCR fragment and triple digested Plasmid DNA RT-PCR Plasmid A260/280 1.93 1.90 Concentration (ng/μl) 6.20 31.8 As seen in Table 3, the A260/280 reading for RT-PCR fragment and Plasmid DNA are 1.93 and 1.90 respectively, indicating that no significant amount of contaminants present. Concentration of RT-PCR fragment is 6.20ng/μl and plasmid DNA is 31.8ng/μl. 3.5 Bacterial Transformation Results Table 4: Transformation results for pET11a with RT-PCR fragment and different ratios LB Plate with Ampicillin Plasmid : RT-PCR Plasmid : RT-PCR 1:1 1:2 Number of Colonies 131 X 8 = 1048 131 X 8 = 1048 In table 4, it can be seen that there were approximately 1048 colonies on both plates with different concentrations of plasmid : RT-PCR mixture.

3.6 Quantitation Results: NanoDrop readings – Plasmid DNA Table 5: Quantitative results of extracted plasmid DNA at ratios from section 3.5 1 : 1 A (A) 1 : 1 B (B) 1 : 2 A (C) A260/280 2.01 2.00 2.05 Concentration (ng/μl) 144.0 130.4 181.3

1 : 2 B (D) 2.01 116.8

In Table 5, it can be seen that all 4 tubes, A, B, C and D have almost the same ratio of A260/280 of approximately 2. Tube A has a ratio of 2.01 which is the same as tube D, tube B has a ratio of 2.00 and tube C has a ratio of 2.05. All tubes were generally free of contaminants. 3.7 Agarose Gel Visualisation – Restriction Enzyme Digestion

Figure 3: Gel Image of Gel Electrophoresis of restriction enzyme digestion products. Lanes 4, 5, 6, 7 contains EcoRI/NdeI double digested plasmid 1, 2, 3, 4 respectively. Lane 1 contains 1kb DNA ladder, lane 2 contains uncut plasmid and lane 3 contains EcoRI digested plasmid 1. In Figure 2, we can see that lane 2, the uncut plasmid, revealed three distinct bands corresponding to nicked at the top nearest to the wells, linear in the middle and supercoiled at the bottom (approximately 4000bp) The rest of the band shows up at around 5000 base pairs, which corresponds to the plasmid. Lanes 4 & 7 shows a second band at around 1000bp, indication successful ligation for EcoRI/NdeI double digested plasmid 1 and 4. Since there is successful ligation for plasmid 1, we would expect to see one band at lane 3, around the 6000bp mark however, the band observed does not seem to be at the 6000bp mark. 3.8 Standard Curve for RT-PCR Table 6: Tabulated Values of Standard Curve for Restriction Enzyme Digestion Band Size (bp) LgmW Distance from well (mm) Distance from gel end (mm) DNA 10000 4.000 13 89 Ladder 4000 3.602 18 89 1500 3.176 28 89 1000 3.000 33 89 750 2.875 37 89

Rf value 0.146067 0.202247 0.314607 0.370787 0.415730

Standard Curve of Restriction Digested Plasmids 4.2 4

4

LogmW

3.8

3.602

3.6 3.4

3.176

3.2

3

3 2.8 2.6

2.875

y = -4.0493x + 4.5044 R² = -0.9763 0.14

0.19

0.24

0.29

0.34

0.39

0.44

Rf

Figure 4: Standard Curve of Restriction Digested Plasmids. LgmW was plotted against Rf with equation y = -4.0493x + 4.5044 and R2 value of -0.9763. The same method (Section 3.3) was used to plot the standard curve of restriction digested plasmids. The calculated values are shown in Table 6 while the standard curve is drawn as shown in Figure 4. Similar to Section 3.5, the R2 value of -0.9736 is negative and close to 1, indicating a strong negative linear correlation between size and the mobility of DNA fragments. Using the same method as in Section 3.5, x = Rf is used to calculate y, and y is then used for calculating 10y to obtain the molecular weight of each band. The three bands in lane 2 is calculated to be 10090, 4847 and 3930. The band at lane three is calculated to be 5382. The remaining 4 lanes all had one band between 17mm and 18 mm which is calculated to be 5121. Lanes 4 & 7 had another band which was calculated to be 911bp which is around the length of the RT-PCR product of the LDHA gene. 3.9 DNA Sequencing Results: 99% Match Table 7: BLAST alignment of sequenced results with Database (Accession Number: NM_010699.2) Query Subject Identity (bp) Identity (%) 1 (pET11a-F) NM_010699.2 974/997 98 2 (LDHA 430-450) NM_010699.2 515/521 99 3 (pET11a-R_ NM_010699.2 907/922 98 4 (LDHA 570-550) NM_010699.2 527/528 99 Aligned NM_010699.2 994/996 99 BLAST was used to align our DNA sequencing results with reference sequence NM_010699.2. As seen in Table 7, the forward and reverse construct of pET11a had a match of 98% and the forward and reverse construct of LDHA had a match of 99% respectively. Our aligned sequence had 99% match with the database and only 2 base pairs were misaligned.

4 Discussion and Conclusion 4.1 Quality of RNA isolation and RT-PCR An absorbance ratio of 260/280 is used to determine the purity of nucleic acids. Generally, a ratio of approximately 1.8 is enough to consider a DNA sample as pure and a ratio of approximately 2.0 to consider RNA samples as pure. From our results in Table 1, our DNA sample in had an absorbance ratio of 1.95 which is accepted as pure but our RNA samples in deviates from the recommended ratio quite

a bit, especially for the case of RNA 1 where there is a deviation of more than 0.3. This suggests that there were contaminants in the RNA sample, which could include DNA or proteins. One possible source of contamination could have occurred during TRIzol® extraction when we were transferring the aqueous phase into a new tube. Even the slightest disturbance of the interphase or organic phase can result in contamination and despite our greatest precautions, it seems like disturbance was still caused. In Table 1, it can also be seen that the concentration for RNA 2 is significantly lower than RNA 1. This could be due to several factors, one of which is the presence of ubiquitous RNases as well as the chemical instability of the RNA itself, resulting in the degradation of RNA. In order to have successful RT-PCR, intact RNA is an essential element (Fleige & Pfaffl, 2006). It is possible that our RNA sample was degraded by RNases in the environment, thus resulting in a lesser than expected concentration In the presence of reverse transcriptase, isolated RNA can be used a template for synthesis of a cDNA strand which can then amplified during PCR. Primers are first used to flank the gene on the RNA strand, then extended with the use of reverse transcriptase. From Figure 1, we can see that only lanes 3 and 5 produced a distinct band around the 1000bp mark, which is expected as these lanes contain RT (+) enzyme cocktail hence RT-PCR can occur. Lanes 4 and 6 did not show any bands as expected as these lanes do not contain reverse transcriptase hence RT-PCR is halted after the annealing of the primer. 4.2 Expected and observed size of RT-PCR product As mentioned above, our RT-PCR product is approximately 1000bp and after calculation in Section 3.3, the actual size was determined to be 991bp. The target gene, LDHA, is approximately 999bp and our RT-PCR product is almost the same, differing by around 8bp only. This slight difference between the cDNA and gDNA could be due to the presence of introns in the gDNA that was not reflected in cDNA. 4.3 Bacterial transformation results As seen in Table 4, we observed that both agar plates colonies present, which is in accordance to our expectations. The presence of the colonies indicates that our bacterial transformation was successful as normal bacterial cells would not be able to survive on the ampicillin plate. The pET11a plasmid contains an ampicillin-resistance gene and if there were any bacterial cells that were unsuccessfully transformed, this agar plate also serves to eliminate them, as these untransformed cells are unable to produce β-lactamase, hence are unable to cleave β-lactam ring of the ampicillin and will not survive. Both plates approximately had 1048 colonies, suggesting that transformation efficiency was similar for both. Of the 1048, it is however unknown if the colonies contained empty or recombinant plasmids. 4.4 DNA isolation and restriction digestion results To determine if the plasmids were empty or recombinant, they were digested with the same two restriction sites EcoRI and NdeI to cut out the LDHA gene from the plasmid. If the plasmid is recombinant, a 1kb band would be observed when gel electrophoresis was conducted. However, if the plasmid was empty, there will not be a 1kb band observed. As seen from Figure 3, lanes 4 and 7 showed a band at the 1kb mark, indicating successful ligation. In lanes 5 and 6, there was no band at the 1kb mark, an indication that the plasmids self-annealed and were transformed into the bacterial cells. Considering that Tube A, which was loaded into lanes 3 and 4, contained recombinant plasmids, the band in lane 3 should have only 1 band around the 6kb mark. All cells in a colony are genetically identical hence when single digestion is conducted, all plasmids should be cut to produce approximately a 6kb fragment. However, the band observed was very thick and seems to span between 5kb and 6kb. There are two possible reasons for this observation. First, the amount of DNA present is too much, resulting in a smear. The second reason is that there might have been a contamination and cells from a second colony might have been scooped up as well and that second colony did not have recombinant plasmids, resulting in a 5kb band that combined with a 6kb band.

In lane 2, three bands were observed that corresponds to the different topoisoforms (nicked, linear and supercoiled. While the calculation tell us that the bands are of different base pairs, all three bands in fact contains DNA of the same length. Both the shape and size of DNA affects the rate of distance migrated and in this case, the shape affected the distance it migrated. Supercoiled plasmids being compact migrated the most while nicked and linear plasmids have open structures thus they move slower. For different ratios of RT-PCR fragment : plasmid, considering out of four plates only two had inserts, the ligation efficiency of 50%. The ligation efficiency was not as high as the ratio of insert to vector was not an ideal ratio of 3:1. When calculating the picomole of ends of each tube with the different ratio, it is observed that for the volume ratio of RT-PCR : Plasmid at 1 : 1, RT-PCR had 0.085pmol ends while the plasmid had 0.085pmol ends. This gives us approximately an insert to vector ratio of 1:1, which is not the ideal ratio. While the volume ratio of RT-PCR : Plasmid at 2 : 1 had a higher insert to vector ratio of 0.11:0.058pmol ends, which roughly translates to a ratio of 2:1, it is still not the ideal ratio. Since the ratio was not ideal, it might explain why only two out of four of our plates had inserts. 4.5 DNA sequencing results All four sequences obtained were compared to the reference sequence and obtained matches of at least 98% as seen in Table 7. Two sequences were made with forward primers and two with reverse primers. To obtain the full sequence of our LDHA gene, these four sequences were aligned and compared to eliminate sequencing errors and the final sequence is shown in Figure A in the appendix. There are two mutations in our gene, one at amino acid 246, converting tyrosine to histidine, and a deletion of the 1391st base pair, resulting in a change of four amino acids, glutamic acid-leucineglutamine-phenylalanine to glycine-cysteine-serine-serine at the end of the gene. The conversion of tyrosine to histidine may be severe as this tyrosine molecule is in the middle of a hydrophobic region (RCSB Protein Data Bank, 2000). This will affect the folding and hence function of the enzyme as histidine is hydrophilic while tyrosine is hydrophobic. Furthermore, the location of this amino acid might be in the active site of the enzyme due to it being in the middle of the gene. The change of four amino acids may not be as severe as it is at the end of the gene. However, coupled with the mutation of tyrosine to histidine, enzymatic function of LDHA might be affected. From this sequencing results, we discovered mutations in our gene that could significantly affect our protein. This suggests that our LDHA gene might not be able to translate our desired protein. While we may not be able to analyse the biochemical aspects of the enzyme, our mutated gene can be used in experiments where knockdown of LDHA genes are required. 4.6 Conclusion In conclusion, while we have been consistently getting results to our expectations, our final sequence of the gene was unexpected as there were two mutations that could render the enzyme inactive. While we may not be able to analyse the aspects of LDHA that work, we are able to analyse our mutated enzyme to determine if mutations are at key sites and how the mutations affect the enzyme in detail. We can then understand how LDHA can be down-regulated to further our research in cancer studies.

Acknowledgements

My group would like to acknowledge our Teaching Assistant, Ho Kung Yu Hilary, for his efforts in guiding us during practical sessions, providing assistance when needed as well as giving us extra information to help us in our studies.

References Fleige, S., M. W. Pfaffl (2006). RNA integrity and the effect on real time qRT-PCR performance. Molecular Aspects of Medicine. 27(2-3), 126-139. Ministry of Health, Singapore. (2015). Principle Causes of Death. Retrieved September 28, from https://www.moh.gov.sg/content/moh_web/home/statistics/Health_Facts_Singapore/Princ ipal_Causes_of_Death.html Rong, Y., W. Wu, X. Ni, T. Kuang, D. Jin, D. Wang, W. Lou (2013). Lactate dehydrogenase A is overexpressed in pancreatic cancer and promotes the growth of pancreatic cancer cells. Tumour Biology. 34(3), 1523-1530. H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, (2000). L-lactate dehydrogenase A chain – P06151 (LDHA_MOUSE). The Protein Data Bank. Nucleic Acids Research, 28, 235-242. Shim, H., C. Dolde, B. C. Lewis, C. S. Wu, G. Dang, R. A. Jungman, R. Dalla-Favera, C. V. Dang (1997). c-Myc transactivation of LDH-A: Implications for tumour metabolism and growth. Proceedings of the National Academy of Sciences of the United States of America. 94(13), 6658-6663 The Jackson Laboratory (2015). Why Mouse Genetics? Retrieved September 29, from http://research.jax.org/mousegenetics/advantages/advantages-of-mouse.html Yao, F., T. Zhao, C. Zhong, Z. Ji, H. Zhao (2012). LDHA is necessary for the tumorigenicity of esophageal squamous cell carcinoma. Tumour Biology. 34(1), 25-31.

Appendix GGCGCTCTACCTTATACGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGACCGTTGAACACCACCAC CGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCGCCATGCCCG CGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGG CGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGA TCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGT TTAACTTTAAGAAGGAGATATACATATGCACCATCACCACCATCATGCAACCCTCAAGGACCAGCTGATTGTG AATCTTCTTAAGGAAGAGCAGGCTCCCCAGAACAAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCTT GTGCCATCAGTATCTTAATGAAGGACTTGGCGGATGAGCTTGCCCTTGTTGACGTCATGGAAGACAAACTCAA GGGCGAGATGATGGATCTCCAGCATGGCAGCCTCTTCCTTAAAACACCAAAAATTGTCTCCAGCAAAGACTAC TGTGTAACTGCGAACTCCAAGCTGGTCATTATCACCGCGGGGGCCCGTCAGCAAGAGGGGGAGAGCCGGCTC AACCTGGTCCAGCGAAACGTGAACATCTTCAAGTTCATCATTCCCAACATTGTCAAGTACAGTCCACACTGCAA GCTGCTGATCGTCTCCAATCCAGTGGATATCTTGACCTACGTGGCTTGGAAAATCAGTGGCTTTCCCAAAAACC GAGTAATTGGAAGTGGTTGCAATCTGGATTCAGCGCGGTTCCGTTACCTGATGGGAGAGAGGCTGGGGGTTC ACGCGCTGAGCTGTCACGGCTGGGTCCTGGGAGAACATGGCGACTCCAGTGTGCCTGTGTGGAGTGGTGTGA ATGTTGCCGGCGTCTCCCTGAAGTCTCTTAACCCAGAACTGGGCACTGACGCAGACAAGGAGCAGTGGAAGG AGGTTCACAAGCAGGTGGTGGACAGTGCCTACGAGGTGATCAAGCTGAAAGGTCACACATCCTGGGCCATTG GCCTCTCTGTGGCAGACTTGGCTGAGAGCATAATGAAGAACCTTAGGCGGGTGCATCCCATTTCCACCATGAT TAAGGGTCTCTATGGAATCAATGAGGATGTCTTCCTCAGTGTCCCATGTATCCTGGGACAAAATGGAATCTCG GATGTTGTGAAGGTGACACTGACTCCTGAGGAAGAGGCCCGCCTGAAGAAGAGCGCAGACACCCTCTGGGG AATCCAGAAGGGCTGCAGTTCTAAGAATTCTGAAGACGAAAGGGCCTCGTGATACGCCCTATTTTTATAGGTT AATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT GTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTG TTTTTGCTCACCCAGAAACGCTGGGGAAAGAAAAGATGCTGAAGATCAGTTGGGTGCACGAGGGGGTTACAT CGAACTGGATCTCAACAGCGGTAGAACCTTGAGAGTTTCGCCCCAAAAACGTTTCCCATGATGAGCCTTTTAA AGTTCTGCTTGTGGCGCGG Figure A: LDHA Forward Contig. ATG: Start Codon TAA: Stop Codon CACCATCACCACCATCAT: Polyhistidine Tag

Range 1: 142 to 1137

Alignment statistics for match #1

Score

Expect

1827 bits(989)

Identities

0.0

CDS: Putative 1

1

Query

409

Gaps

994/996(99%) A

T

L

K

D

Q

L

I

Strand

1/996(0%) V

N

L

L

K

E

Plus/Plus E

Q

A

P

Q

N

GCAACCCTCAAGGACCAGCTGATTGTGAATCTTCTTAAGGAAGAGCAGGCTCCCCAGAAC

468

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

142

CDS:L-lactate dehydr

2

GCAACCCTCAAGGACCAGCTGATTGTGAATCTTCTTAAGGAAGAGCAGGCTCCCCAGAAC A

T

L

K

D

Q

L

I

V

N

L

L

K

E

E

Q

A

P

Q

N

CDS: Putative 1

21

K

I

T

V

V

G

V

G

A

V

G

M

A

C

A

I

S

I

L

M

Query

469

AAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCTTGTGCCATCAGTATCTTAATG

201

528

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

202

CDS:L-lactate dehydr

22

AAGATTACAGTTGTTGGGGTTGGTGCTGTTGGCATGGCTTGTGCCATCAGTATCTTAATG K

I

T

V

V

G

V

G

A

V

G

M

A

C

A

I

S

I

L

M

261

CDS: Putative 1

41

Query

529

Sbjct

262

CDS:L-lactate dehydr

42

CDS: Putative 1

61

Query

589

K

D

L

A

D

E

L

A

L

V

D

V

M

E

D

K

L

K

G

E

AAGGACTTGGCGGATGAGCTTGCCCTTGTTGACGTCATGGAAGACAAACTCAAGGGCGAG

588

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAGGACTTGGCGGATGAGCTTGCCCTTGTTGACGTCATGGAAGACAAACTCAAGGGCGAG K

D

L

A

D

E

L

A

L

V

D

V

M

E

D

K

L

K

G

E

M

M

D

L

Q

H

G

S

L

F

L

K

T

P

K

I

V

S

S

K

ATGATGGATCTCCAGCATGGCAGCCTCTTCCTTAAAACACCAAAAATTGTCTCCAGCAAA

321

648

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

322

ATGATGGATCTCCAGCATGGCAGCCTCTTCCTTAAAACACCAAAAATTGTCTCCAGCAAA

CDS:L-lactate dehydr

62

M

M

D

L

Q

H

G

S

L

F

L

K

T

P

K

I

V

S

S

K

CDS: Putative 1

81

D

Y

C

V

T

A

N

S

K

L

V

I

I

T

A

G

A

R

Q

Q

Query

649

Sbjct

382

CDS:L-lactate dehydr

82

D

Y

C

V

T

A

N

S

K

L

V

I

I

T

A

G

A

R

Q

Q

CDS: Putative 1

101

E

G

E

S

R

L

N

L

V

Q

R

N

V

N

I

F

K

F

I

I

Query

709

GACTACTGTGTAACTGCGAACTCCAAGCTGGTCATTATCACCGCGGGGGCCCGTCAGCAA

381

708

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GACTACTGTGTAACTGCGAACTCCAAGCTGGTCATTATCACCGCGGGGGCCCGTCAGCAA

GAGGGGGAGAGCCGGCTCAACCTGGTCCAGCGAAACGTGAACATCTTCAAGTTCATCATT

441

768

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

442

GAGGGGGAGAGCCGGCTCAACCTGGTCCAGCGAAACGTGAACATCTTCAAGTTCATCATT

CDS:L-lactate dehydr

102

E

G

E

S

R

L

N

L

V

Q

R

N

V

N

I

F

K

F

I

I

CDS: Putative 1

121

P

N

I

V

K

Y

S

P

H

C

K

L

L

I

V

S

N

P

V

D

Query

769

CCCAACATTGTCAAGTACAGTCCACACTGCAAGCTGCTGATCGTCTCCAATCCAGTGGAT

501

828

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

502

CDS:L-lactate dehydr

122

CCCAACATTGTCAAGTACAGTCCACACTGCAAGCTGCTGATCGTCTCCAATCCAGTGGAT P

N

I

V

K

Y

S

P

H

C

K

L

L

I

V

S

N

P

V

D

CDS: Putative 1

141

I

L

T

Y

V

A

W

K

I

S

G

F

P

K

N

R

V

I

G

S

Query

829

ATCTTGACCTACGTGGCTTGGAAAATCAGTGGCTTTCCCAAAAACCGAGTAATTGGAAGT

561

888

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

562

CDS:L-lactate dehydr

142

CDS: Putative 1

161

Query

889

ATCTTGACCTACGTGGCTTGGAAAATCAGTGGCTTTCCCAAAAACCGAGTAATTGGAAGT I

L

T

Y

V

A

W

K

I

S

G

F

P

K

N

R

V

I

G

S

G

C

N

L

D

S

A

R

F

R

Y

L

M

G

E

R

L

G

V

H

GGTTGCAATCTGGATTCAGCGCGGTTCCGTTACCTGATGGGAGAGAGGCTGGGGGTTCAC

621

948

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

622

GGTTGCAATCTGGATTCAGCGCGGTTCCGTTACCTGATGGGAGAGAGGCTGGGGGTTCAC

CDS:L-lactate dehydr

162

G

C

N

L

D

S

A

R

F

R

Y

L

M

G

E

R

L

G

V

H

CDS: Putative 1

181

A

L

S

C

H

G

W

V

L

G

E

H

G

D

S

S

V

P

V

W

Query

949

GCGCTGAGCTGTCACGGCTGGGTCCTGGGAGAACATGGCGACTCCAGTGTGCCTGTGTGG ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

681

1008

Sbjct

682

CDS:L-lactate dehydr

182

CDS: Putative 1

201

Query

1009

GCGCTGAGCTGTCACGGCTGGGTCCTGGGAGAACATGGCGACTCCAGTGTGCCTGTGTGG A

L

S

C

H

G

W

V

L

G

E

H

G

D

S

S

V

P

V

W

S

G

V

N

V

A

G

V

S

L

K

S

L

N

P

E

L

G

T

D

AGTGGTGTGAATGTTGCCGGCGTCTCCCTGAAGTCTCTTAACCCAGAACTGGGCACTGAC

741

1068

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

742

AGTGGTGTGAATGTTGCCGGCGTCTCCCTGAAGTCTCTTAACCCAGAACTGGGCACTGAC

CDS:L-lactate dehydr

202

S

G

V

N

V

A

G

V

S

L

K

S

L

N

P

E

L

G

T

D

CDS: Putative 1

221

A

D

K

E

Q

W

K

E

V

H

K

Q

V

V

D

S

A

Y

E

V

Query

1069

Sbjct

802

CDS:L-lactate dehydr

222

CDS: Putative 1

241

Query

1129

GCAGACAAGGAGCAGTGGAAGGAGGTTCACAAGCAGGTGGTGGACAGTGCCTACGAGGTG

801

1128

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GCAGACAAGGAGCAGTGGAAGGAGGTTCACAAGCAGGTGGTGGACAGTGCCTACGAGGTG A

D

K

E

Q

W

K

E

V

H

K

Q

V

V

D

S

A

Y

E

V

I

K

L

K

G

H

T

S

W

A

I

G

L

S

V

A

D

L

A

E

ATCAAGCTGAAAGGTCACACATCCTGGGCCATTGGCCTCTCTGTGGCAGACTTGGCTGAG

861

1188

||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||| Sbjct

862

ATCAAGCTGAAAGGTTACACATCCTGGGCCATTGGCCTCTCTGTGGCAGACTTGGCTGAG

CDS:L-lactate dehydr

242

I

K

L

K

G

Y

T

S

W

A

I

G

L

S

V

A

D

L

A

E

CDS: Putative 1

261

S

I

M

K

N

L

R

R

V

H

P

I

S

T

M

I

K

G

L

Y

Query

1189

AGCATAATGAAGAACCTTAGGCGGGTGCATCCCATTTCCACCATGATTAAGGGTCTCTAT

921

1248

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

922

CDS:L-lactate dehydr

262

AGCATAATGAAGAACCTTAGGCGGGTGCATCCCATTTCCACCATGATTAAGGGTCTCTAT S

I

M

K

N

L

R

R

V

H

P

I

S

T

M

I

K

G

L

Y

CDS: Putative 1

281

G

I

N

E

D

V

F

L

S

V

P

C

I

L

G

Q

N

G

I

S

Query

1249

GGAATCAATGAGGATGTCTTCCTCAGTGTCCCATGTATCCTGGGACAAAATGGAATCTCG

981

1308

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

982

GGAATCAATGAGGATGTCTTCCTCAGTGTCCCATGTATCCTGGGACAAAATGGAATCTCG

CDS:L-lactate dehydr

282

G

I

N

E

D

V

F

L

S

V

P

C

I

L

G

Q

N

G

I

S

CDS: Putative 1

301

D

V

V

K

V

T

L

T

P

E

E

E

A

R

L

K

K

S

A

D

Query

1309

GATGTTGTGAAGGTGACACTGACTCCTGAGGAAGAGGCCCGCCTGAAGAAGAGCGCAGAC

1041

1368

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct

1042

GATGTTGTGAAGGTGACACTGACTCCTGAGGAAGAGGCCCGCCTGAAGAAGAGCGCAGAC

CDS:L-lactate dehydr

302

D

V

V

K

V

T

L

CDS: Putative 1

321

T

L

W

G

I

Q

K

Query

1369

Sbjct

1102

CDS:L-lactate dehydr

322

T

G

P

C

E

S

E

E

A

R

L

K

K

S

A

1101

D

S

ACCCTCTGGGGAATCCAGAAGG-GCTGCAGTTCTAA

1403

|||||||||||||||||||||| ||||||||||||| ACCCTCTGGGGAATCCAGAAGGAGCTGCAGTTCTAA T

L

W

G

I

Q

K

E

L

Q

1137

F

Figure B: Alignment between forward contig and database for LDHA gene. Highlighted in blue are mutations.