Examining Transgenic Animals And Treatments Biology Essay
pIns-c-MycERTAM was generated by cloning a full-length human c-myc cDNA fused to the hormone-binding domain of a modified estrogen receptor (c-MycERTAM) downstream of the rat insulin promoter as previously described (Pelengaris, Khan et al. 2002). DNA constructs were injected into male pronuclei of day 1-fertilized (CBA × C57BL/6) F1 embryos and injected embryos were transferred into day 1-plugged pseudopregnant foster mice and the litters screened for presence of the transgene by Southern blotting. Heterozygous founder mice were backcrossed appropriately to establish transgenic lines.
IGF-II knockout strain was obtained from Dr. Douglas Hanahan (Christofori, Naik et al. 1994). As mentioned in Chapter 1.2.1, igf2 is an imprinted gene with maternally inherited allele silenced. Thus by crossing with female pIns-cMycERTAM mice we created the pIns-cMycERTAM/IGF-II+/+ (IGF-II wildtype) mice and pIns-cMycERTAM/IGF-II+/- (IGF-II KO) mice.
PML KO strain was obtained and maintained from Dr. Keith Leppard (Leppard, Emmott et al. 2009), originally generated by Dr. Pandolfi’s group (Wang, Delva et al. 1998). Heterozygous PML+/- were selected to breed with pIns-cMycERTAM mice to created pIns-cMycERTAM/PML+/+ (PML wildtype) and pIns-cMycERTAM/PML-/- (PML KO).
Heterozygous RIP7-Bcl-xL transgenic mice were obtained from Dr. Doug Hanahan (UCSF) (Zhou, Pena et al. 2000) and selected to cross with pIns-cMycERTAM mice. Offspring genotyped as RIP7-Bcl-xL/pIns-cMycERTAM were crossed with PML+/- single strain to created RIP7-Bcl-xL/pIns-cMycERTAM /PML+/+ (RIP/PML wildtype) and RIP7-Bcl-xL/pIns-cMycERTAM/PML-/- (RIP/PML KO).
Transgenic mice were housed under barrier conditions with a 12 hour light/dark cycle and access to food and water.
2.2 Treatment of Transgenic Animals
2.2.1 Genotyping Transgenic Animals
Litters from all transgenic mice and appropriate F1 crosses were routinely genotyped by PCR analysis on DNA (1 to 5μl) isolated from ear biopsies. DNA was extracted by incubating each ear disc in 75μl "Hotshot" reagent (25mM NaOH, 0.2mM disodium ethylenediaminetetra acetic acid (EDTA) and pH12) for 30 minutes at 95°C. Following this, an equal volume of neutralizing agent (40mM Tris-HCl, pH5) was added and the sample was cooled to 4°C overnight. DNA samples were stored at -20°C for long-term storage
Primers used for the detection of c-MycERTAM DNA: (forward) 5' CCA AAG GTT GGC AGC CCT CAT GTC 3'; (reverse) 5' AGG GTC AAG TTG GAC AGT GTC AGA GTC 3'. Reagents for PCR: DNA 3μl, H2O 17.25μl, X10 PCR Buffer (Invitrogen, Carlsbad, CA) 2.5μl, MgCl2 (50mM Invitrogen, Carlsbad, CA) 0.75μl, MYC’5 (Forward Primer; 10pmol/μl) 0.5μl, MERTM (Reverse Primer; 10pmol/μl) 0.5μl, deoxynucleotide triphosphate (dNTPs; 10mM; Invitrogen, Carlsbad, CA) 0.25μl and Taq polymerase (5U/μl; Invitrogen, Carlsbad, CA) 0.25μl.
PCR program: 94°C 2 min 1 cycle, [94°C 1 min, 57°C 1 min, 72°C 2 min] 30 cycles, 72°C 10 min 1 cycle.
Primers used for the detection of IGF-II KO DNA: (forward) 5' GAT GGA TTG CAC GCA GGT TC 3'; (reverse) 5' CTT CAG TGA CAA CGT CGA GC 3'. PCR reagents: DNA 3μl, H2O 17.25μl, X10 PCR Buffer (Invitrogen, Carlsbad, CA) 2.5μl, MgCl2 (50mM, Invitrogen, Carlsbad, CA) 0.75μl, NeoIGF2F (Forward Primer; 10pmol/μl) 0.5μl, NeoIGF2R (Reverse Primer; 10pmol/μl) 0.5μl, deoxynucleotide triphosphate (dNTPs; 10mM; Invitrogen, Carlsbad, CA) 0.25μl and Taq polymerase (5U/μl; Invitrogen, Carlsbad, CA) 0.25μl.
PCR program: 94°C 2 min 1 cycle, [94°C 30s, 62°C 1 min, 72°C 1 min] 30 cycles, 72°C 3 min 1 cycle.
Primers used for the detection of PML DNA: (forward 1) 5' TTT CAG TTT CTG CGC TGC 3'; (forward 2) 5' CGA CCA CCA AGC GAA ACA 3' (reverse) 5' TTG GAC TTG CGC GTA CTG TC 3'. PCR reagents: DNA 3μl, H2O 15.85μl, X10 PCR Buffer (Invitrogen, Carlsbad, CA) 2.5μl, MgCl2 (50mM, Invitrogen, Carlsbad, CA) 0.75μl, PML F (Mix Froward Primer 1 and 2, 10pmol/μl) 0.5μl, PML R (Reverse Primer; 10pmol/μl) 0.5μl, deoxynucleotide triphosphate (dNTPs; 10mM; Invitrogen, Carlsbad, CA) 0.5μl and Taq polymerase (5U/μl; Invitrogen, Carlsbad, CA) 0.4μl.
PCR program: 95°C 10 min 1 cycle, [94°C 1 min, 57°C 1 min, 72°C 1 min] 35 cycles, 72°C 10 min 1 cycle.
Primers used for the detection of RIP7-Bcl-xL DNA: (forward) 5' CAG CTC CCG GTT GCT CTG AGA C 3’; (reverse) 5’AGC ACT TTC TGC AGA CCT AGC AC 3’ PCR reagents (Initrogen Taq kit): DNA 3μl, H2O 15.6μl, X10 PCR Buffer 2.5μl, MgCl2 (50mM, Invitrogen, Carlsbad, CA) 1.5μl, MBD-XF (Froward Primer; 10pmol/μl) 1μl, RIP-2 (Reverse Primer; 10pmol/μl) 1μl, deoxynucleotide triphosphate (dNTPs; 10mM; Invitrogen, Carlsbad, CA) 0.2μl and Taq polymerase (5U/μl; Invitrogen, Carlsbad, CA) 0.2μl.
PCR program: 94°C 1 min 1 cycle [94°C 1 min, 60°C 30s, 72°C 2 min] 30 cycles, 72°C 3 min 1 cycle.
PCR products, positive and negative control samples (MycERTAM-positive, IGF-II KO-positive, PML KO/heterozygous-positive, RIP7-BcL-XL-positvie DNA, wildtype DNA and water respectively) and a 1kb DNA ladder mix (Invitrogen, Carlsbad, CA; 10μl ladder mix contains 9μl dH2O and 1μl DNA ladder (1μg/μl)) were loaded on a 1% agarose gel with Tris/borate/EDTA buffer (TBE; 0.89M Tris base, 0.02M EDTA-Na2-salt, 0.89M boric acid) and with a 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol in water at 4°C). The gel was run for 2-3 hours at 100 V and migration was captured using the Gene Genius Bio Imaging System with the GeneSnap Image Capture Suite (Syngene, Frederick, MD).
2.2.2 Administration of 4-hydroxytamoxifen (4-OHT)
To activate c-MycERTAM protein in pancreatic beta cells of adult transgenic mice, 1mg of 4-hyfroxytamoxifen (4-OHT; Sigma-Aldrich, St. Louis, MO, T7904) sonicated in peanut oil (1mg/0.2ml) was administered daily by intraperitoneal injection. Inactivation of c-MycERTAM protein was achieved following withdrawal of 4-OHT (Pelengaris, Khan et al. 2002). Control mice received equal volumes of their vehicle (peanut oil) without 4-OHT daily. Alternatively, in some experiments Tamoxifen (Sigma-Aldrich, St. Louis, MO, T5648) was used to activate c-MycERTAM transgene (Cano, Rulifson et al. 2008; Radziszewska, Choi et al. 2009). Blood glucose was monitored for each mouse in experiments and recorded for the indication of diabetes or recovery.
2.2.3 Administration of Bromodeoxyuridine (BrdU)
5-bromo-2-deoxyuridine (5-BrdU) is an analogue of thymidine and commonly used in detecting proliferating cells. By substituting thymidine during DNA replication BrdU can be incorporated into the newly synthesized DNA of replicating cells, specifically the ones in the S phase of the cell cycle. In our study at the end of some experiments 2μmol 5-BrdU (Sigma-Aldrich, St. Louis, MO, B5002) was administrated by intraperitoneal injection 3.5hr prior to sacrifice (Finch, Prescott et al. 2006). Antibodies specific for BrdU were used in detecting beta cell proliferation rate quantification.
2.2.4 Intraperitoneal Glucose Tolerance Test (IPGTT)
Animals were injected intraperitoneally with 30% D-glucose in dH2O (1.5mg/g body weight) after overnight starvation (16 hours). 2 to 3μl of blood were collected from the tip of the animals’ tail to monitor the blood glucose levels prior to, 20, 60 and 120 minutes after glucose injection by Advantage glucose meter (Roche Diagnostics). Wildtpye mice were also performed IPGTT considered as controls.
2.2.5 Pancreas Excision and Preparation
Pancreata were dissected immediately after sacrifice. To make frozen section Pancreata were fixed in 4% paraformaldehyde (PFA) for 2hr, transferred to 30% sucrose in phosphate buffer saline (PBS) overnight at 4°C, embedded in OCT, and frozen in foil on a bath of dry ice and ethanol (70%) and store in -80°C. Also respectively tissues were fixed overnight in neutral-buffered formalin, embedded in paraffin wax for the preparation of paraffin section. In the RNA/protein assays pancreata were immediately frozen in liquid nitrogen after dissection. Tissue samples were stored at -80 °C for long term storage.
2.3 RNA Extracting and Reverse Transcription
Total RNA was isolated from homogenised pancreata using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA) for small sample RNA preparation, incorporating a deoxyribonuclease (DNase) I treatment step to remove DNA molecules. RNA quantity and quality were tested by Nanodrop spectrophotometer to estimate the concentration. RNA integrity was protected by cleaning work areas and instruments with RNaseZap® (Ambion, Austin, TX) and DEPC-treated water to inactivate RNases.
Total pancreas RNA was isolated and stabilized by homogenizing the pancreas in 2ml RLT buffer (with 20μl (100:1) β-mecaptolethonal) for 1 minute (For islets RNA extraction 1ml RLT buffer (with 10μl (100:1) β-mecaptolethonal) were used in homogenized by pippetting). After homogenizing samples stood in ice for 5 minutes to precipitate the impurities and 1 volume (650μl) of sample stock was taken and added by 1 volume (650μl) of 70% ethanol to the homogenized lysate and mixed thoroughly by pipetting. The mixture was added to an RNeasy MinElute silica-gel column (Qiagen, Valencia, CA) in a 2ml collection tube and centrifuged at full speed for 15 seconds (4°C). The flow-through was discarded, and 350μl buffer RW1 (a guanidinium thiocyanate-containing buffer; Qiagen, Valencia, CA) was added to the spin column before centrifuging at full speed for 15 seconds to wash the column (4°C). The column was washed by adding 350μl buffer RW1 and centrifuging for 15 seconds at full speed. The MinElute 136 column was transferred into a new 2ml collection tube, 500μl buffer RPE (Qiagen, Valencia, CA), diluted from fresh in 4 volumes 80% ethanol, was added to the column and centrifuged for 2 minutes at full speed to dry the silica-gel membrane (4°C). The MinElute column was transferred to a new 2ml collection tube and centrifuged with an open cap at full speed for 5 minutes to further dry the membrane. RNA was eluted by adding 30μl nuclease-free water directly onto the silica-gel membrane and centrifuging at full speed for 1 minute (4°C). RNA extracted and purified were stored at -80°C for long term usage.
To remove genomic DNA from extracted RNA samples, DNase I incubation mix (10μl DNase I stock solution, 70μl buffer RDD (Qiagen, Valencia, CA) was applied directly to RNA samples for 10 minutes at RT. And the samples were performed “RNA Clean Up”, which is the same procedure as mentioned above.
2.4 Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
2.4.1 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Extracted and purified RNA of each pancreas was immediately processed to reverse transcription polymerase chain reaction (RT-PCR). Total RNA of 400ng from each sample were used in RT-PCR. Briefly for 1μl RNA sample (400ng/μl) 0.4μl RT random oligo (dT) primers was added and adjust the volume to 11μl by water. And samples were incubated in 70°C for 5 minutes and quickly chilled on ice. Then each sample was spin down and added master mix (5X First-Strand Buffer 4μl, 0.1M DTT 2μl, 10mM dNTP 1μl and dH2O 1μl). Samples were incubated under 25°C for 5 minutes and added 1μl (200 units) of SuperScript™ II RT (reverse transcriptase; Invitrogen Carlsbad, CA) Samples then were incubated in 42°C for 60 minutes and 70°C for 10 minutes. cDNA was stored at 4°C for short term storage (up to 24 hours) or at -20°C for long term storage.
2.4.2 IGF-II cDNA PCR and Product Purification
IGF-II cDNA was used to perform general PCR for the detection of IGF-II expression. Following primers were used: IGF2F: 3’- GTG CGG AGG GGA GCT TGT TGA C-5’; IGF2R: 5’-GTG GGC GTC TTT GGG TGG TA-3’. PCR reagents (Initrogen Taq Kit): DNA 3μl, H2O 17.25μl, PCR Buffer 2.5μl, MgCl2 0.75μl, IGF2F (Forward Primer; 10pmol/μl) 0.5μl, IGF2R (Reverse Primer; 10pmol/μl) 0.5μl, dNTPs (10mM; Invitrogen, Carlsbad, CA) 0.25μl and Taq polymerase (5U/μl; Invitrogen, Carlsbad, CA) 0.25μl. PCR program: 94°C 2 min 1 cycle, [94°C 1 min, 57°C 1 min, 72°C 2 min] 40 cycles, 72°C 10 min 1 cycle. PCR product size: 357 bp
IGF-II PCR products, including positive and negative control samples (mouse E18.5 placenta cDNA, and water respectively) and a 1kb DNA ladder mix (Invitrogen, Carlsbad, CA; 10μl ladder mix contains 9μl dH2O and 1μl DNA ladder (1μg/μl)) were loaded on a 1% agarose gel with Tris/borate/EDTA buffer (TBE; 0.89M Tris base, 0.02M EDTA-Na2-salt, 0.89M boric acid) and with a 6x loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF and 30% glycerol in water at 4°C). The gel was run for 3.5 hours at 90V and migration was captured using the Gene Genius Bio Imaging System with the GeneSnap Image Capture Suite (Syngene, Frederick, MD).
PCR product purification was performed by using QIAquick Gel Extraction Kit (Qiagen). Target bands from gel were isolated and PCR products were purified for sequencing. Briefly PCR bands were excised from gel and added 3 volumes of Buffer PB to 1 volume of the PCR sample and incubate at 50°C for 10 minutes. After the gel slice has dissolved completely 1 gel volume of isopropanol was added to the sample and mix. Then samples were applied to the QIAquick column and centrifuge for 60 seconds. Flow-through was discarded and samples were added 0.75ml Buffer PE to the QIAquick column and centrifuge for 60 seconds to wash. Flow-through was discarded and samples were centrifuged in a new column for an additional 60 seconds. 30μl elution buffer was added to the center of the QIAquick membrane, and stand for 1 minute, and then centrifuge at full speed to harvest DNA. The purified cDNA PCR product was sequenced by Molecular Biology Service, Biological Department, University of Warwick.
2.4.3 Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
qRT-PCR was set up on a 96-well PCR plate. For each well, the PCR master mix consisted of 10μl TaqMan 2X gene-expression Master Mix, 9μl cDNA sample and 1μl TaqMan qRT-PCR gene-expression assay or 18s rRNA control. Individual gene-expression assays, 18s rRNA endogenous positive control probe (Applied Biosystems, Foster City, CA) and water negative control were run in triplicate wells. qRT-PCR was performed using an ABI Prism 7000 scanner (Applied Biosystems, Foster City, CA), using the following program: 2 min at 50°C for activation of Uracil-DNA glycosylase, 10 minutes at 95°C for activation of AmpliTaq Gold enzyme and 40 cycles of 95°C for 15 seconds (denature) and 60 °C for 1 minute (anneal/extension).
2.4.4 Analysis of qRT-PCR Data
One approach typically used in gene expression quantification is comparative CT method. Briefly in our qRT-PCR essay the fluorescence probe binds to the transcript of interest and fluorescence levels increase following each PCR cycle in proportion to the amount of amplicon produced in the PCR reaction. A threshold value for signal detection is set, and the (fractional) cycle number at which fluorescence levels first exceed this value is termed the threshold cycle (CT). This value is used to estimate transcript abundance. A smaller CT value indicates higher transcript abundance in the original RNA sample. In the experiment by normalizing CT of the target gene (igf2 in this case) with CT of the endogenous control 18s rRNA (which should be equally expressed in all cells) and CT of experiment controls (which should be RNA from mice without c-Myc activation), relative quantity was of igf2 reexpression following c-Myc induced beta cell injury was determined.
The detail of calculation was given by:
The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by:
Relative Igf2 expression quantity = 2-4
2.5 Pancreatic Islet Isolation
2.5.1 Solution Preparation
HBSS1 medium is made with 500ml HBSS (Hanks Balanced Salt Solution) and added 2.4ml of NaHCO3 at 7.5%, 10ml of HEPES at 1M pH 7.4, 1.4ml of mixture antibiotics containing Penicillin (10000IU/ml) and Streptomycin (10mg/ml). HBSS1-10% FCS medium is made with HBSS1 plus 10% (v/v) of foetal calf serum (FCS, Heat Inactivated; Sigma-Aldrich, St. Louis, MO).
RPMI1 medium is made with 500mls RPMI 1640 pH7.4, 0.9g of glucose (final concentration at 10mM), 1.5ml of the same antibiotic solution cited above, BSA (bovine serum albumin) at 5g/l and 5ml of L-glutamine.
Collagenase solution is made with 9ml of HBSS1 medium and 10mg of collagenase (Invitrogen) with protease inhibitor (1mg/ml, Type V, Sigma-Aldrich)
2.5.2 Islet Isolation
Pancreata from mouse were injected with 2.5ml Collagenase and added with 10ml HBSS1 (pre-warmed in 35°C water bath) and digested in 35°C water bath for 15 minutes. Then tubes were shaken by hand for 1 minute and added 10ml cold HBSS1 to stop the digestion. After 3 times wash in HBSS1-10% FCS islets were transfer to Petri dish incubated with RPMI1. And islets were picked up by hand under light microscope and washed in cold PBS for 2 times. Clean islets were stored -80°C for long term storage. Further step of RNA extraction and RT-PCR were performed as described in Chapter 2.3 and 2.4.
2.6 Immunohistochemical Staining of Tissue
2.6.1 Haematoxylin & Eosin Staining
Frozen OCT-embedded tissue sections were cut to 10μm using a Bright 5040 cryostat (Jencons Scientific, Bridgeville, PA) and sequentially washed in Haematoxylin solution (Surgipath, Richmond, IL) for 135 seconds, tap water for 45 seconds, acid alcohol solution (70% ethanol, 0.5% HCl) for 45 seconds, tap water for 45 seconds, Scott’s solution (40g MgSO4, 7g NaHCO3 and Thymol crystals in 2L tap water), 1% aqueous Eosin solution (VWR International Ltd., Leicestershire, England) for 135 seconds and tap water for 45 seconds. Sections were dried by sequential washing in 4 ethanol solutions graded from 50% to 95% for 1 minute each and 2 changes in 100% for 1 minute each. Finally sections were washed in two changes of xylene for 5 minutes each. Sections were mounted immediately in p-xylene-bis-pyridinium bromide (DPX) mounting medium (Agar Scientific Ltd., Essex, England). Paraffin embedded tissue sections were first incubated in oven at 60°C for 40 minutes and washed in two changes of xylene for 10 minutes each, following wash two time in 100% ethanol for 1 minute each. And the sections were sequentially washed in 4 ethanol solutions graded from 95% to 50% for 1 minute each and carried on with the same protocol as used in frozen section. Sections were viewed using an Axiostar Plus light microscope (Zeis, Oberkochen, Germany). Images were captured using a Powershot G5 digital camera (Canon, Tokyo, Japan).
2.6.2 Immunofluorescence Staining
Sections were blocked by 10% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, A-7906) for 30 minutes at room temperature (RT) or goat serum dilution (150μl goat serum (Sigma-Aldrich, St. Louis, MO, G9023), 50μl Triton X-100, 10ml PBS) for 1 hour at RT and then incubated with primary antibodies. Antibodies used in the experiment were listed as following: Guinea pig anti-swine Insulin (1:100 dilutions; Dako), Rabbit anti-Human Glucagon (1:100 dilutions; Dako), Rabbit anti-Human Ki67 (1:200 dilutions; Novocastra, UK) and Sheep polyclonal to BrdU antibody (1:100 dilutions; Abcam, AB1893). Following fluorescence probes were used: Alexa 633 goat anti-Guinea pig (1:200 dilutions; Invitrogen), Fluorescein (FITC) goat anti-Rabbit IgG (H+L) (1:200 dilutions; Vector), FITC Donkey Anti-Sheep IgG (H+L) (1:200 dilution; Jacksons Lab, 713-095-147) and 4', 6-diamidino-2-phenylindole (DAPI; Vector H-1200). Fluoresces stained slides were observed under confocal microscopy (Leica SP2).
2.7 Quantification Analysis of Pancreas
At least five slides (stained with insulin and DAPI) from each animal were scanned under confocal microscopy and images were captured by scanning each whole section (whole pancreas under magnification of 50X and islets captured under 400X). Software Image J (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/) was used for calculating insulin and DAPI stains areas. Beta cell mass was calculated by achieving insulin staining percentage and multiply by the pancreas weight (Bonner-Weir 2001). Moreover total beta cell number from same sections was counted manually as well as by software. Such data were also normalized by total pancreas area from each section. To avoid any bias, confocal capture and software analysis of insulin/pancreas area or beta cell number were carried out blindly.
Results between groups were compared by Student t test, with a p value < 0.05 considered as achieving statistical significance. All results were reported as the means± standard error of the mean (SEM).
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