Mir151 conventional knockout mice
6.3. Research motive and the strategy
The discovery of miRNAs as modifiers of disease processes can help identify
cellular effectors and define molecular mechanisms of disease processes. Unlike the
widely studied miRNAs, miR-151-associated reports is limited thus the physiological
and pathological role of MIR151 remains extremely unclear, and a genetic knockout
mice may provide an opportunity to investigate the function of MIR151. In this study,
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we established the conventional knockout mice lack of Mir151 in the attempt to
investigate the physiological function of Mir151, and whether Mir151 participates in
cancer biology.
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2x TaqMan Universal PCR Master Mix Applied Biosystems
Agar, Bacteriological ALPHA biosciences
Agarose Invitrogen
Ampicillin Sigma
Boric acid J.T.Baker
BPB Sigma
Calf Intestine Alkaline Phosphatase (CIP) Fermentus
Chlorofrom J.T.Baker
Phosphate buffered saline (PBS) Biowest
T-PER buffer Thermo
Tris base J.T.Baker
Tryptone ALPHA biosciences
Urethane Sigma
Yeast extract ALPHA biosciences
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7.1.2. Kits
Kit Company
FastStart universal SYBR green master (ROX) Roche
FavorPrep plasmid extraction midi kit FAVORGEN FavorPrep plasmid extraction mini kit FAVORGEN
Qiagen Plasmid Maxi kit Qiagen
RnaseOut Invitrogen
SuperScript III Reverse Transcriptase Invitrogen
T4 DNA Ligase NEB
Taq DNA polymerase Geneaid
TaqMan miRNA expression assay Applied Biosystems
TaqMan® miRNA RT kit Ambion
7300 Real-Time PCR machine Appied Biosystems
Abbott Cell-Dyn 3700 GMI
AllegraTM 21R centriguge Beckman Coulter
Analytical balance (TE124s) Sartorius
Avanti® J-E high speed centrifuge Beckman Coulter Eppendorf microcentriguge (F45-24-11) Eppendorf
GeneAmp PCR machine Appied Biosystems
Hitachi 7170A Automatic Analyzer HITACHI
hypoxia chamber homemade
LAB ROTATOR Digisystem
Nanodrop Thermo
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Orbital shacking incubator (OSI500R) TKS
7.2. Methods
7.2.1. Targeting vector construction
The recombineering technology (20) was used in the construction of targeting vector for
Mir151 deletion (Appendix VII). BAC (Bacterial artificial clone) carrying isogenic
genomic DNA of Mir151 (bMQ-355P20) under the 129sv/J genetic background was
purchased from Source BioScience LifeSciences. The sequences used for homologous
recombination were amplified from the BAC using PCR (AB and YZ fragment, see
Appendix VIII) and then clone into pL253 vector, called “Vector A”. Vector A was
linearized by restriction enzyme digestion and co-transformed with bMQ-355P20 BAC
into EL350 E.coli by electroporation. Vector A which retrieved the chromosomal
fragment from BAC through homologous recombination was called “Retrieve A”. To
insert the loxP sequences into the flanked regions of Mir151 gene, homologous DNA
fragment CD/EF and GH/IJ were cloned into pL452 and pL451 vector, called “Vector B”
and “Vector C”, respectively (Appendix VIII). “A+B” was produced by transforming
the “Retrieve A” and “Vector B” together into EL350 E.coli and the loxP-Neor-loxP
sequence originated form Vector B would integrate into the 3ʹ end of Mir151 through
homologous recombination. Next, the addiction of Arabinose induced Cre recombinase
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expression in EL350 E.coli and deleted one of the loxP site and Neor, called “A+B-B”.
With similar principle, the introduction of Frt-PGK-Neor-Frt-loxP sequence from
“Vector C” into 5ʹ end which called “A+B-B+C” was performed in the next. Finally,
after linearized the “retrieve A+B-B+C” with Not I restriction enzyme, the targeting
vector containing neomycin resistance gene and thymidine kinase for positive and
negative selection in ES cell culture was ready for ES cell targeting. The primer
sequences used for targeting vector construction were shown in Table 1.
7.2.2. Gene targeting of ES cells and generation of Mir151 conventional knockout (KO) mice
The E14TG2a (HPRT-) ES cell line was cultured, propagated, and transfected with the
targeting construct by electroporation. HAT (0.1mM hypoxanthine, 4μM aminopterin,
and 0.16 mM thymidine) and ganciclovir (10 μM) was used to select for ES cell
colonies grown from electroporation. Surviving cell colonies were isolated, established
as clones, and genotyped by Southern blotting to ensure homologous recombination.
Southern blotting and genomic DNA isolation were performed following standard
procedures to identify the desired ES cell clones. The correct clones were subsequently
introduced into blastocysts of C57BL/6J mice by microinjection. Chimeric mice were
bred with wild-type (WT) C57BL/6J mice to obtain heterozygous first generation
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(B6129-N1F1) mice, which were intercrossed to generate homozygous N1F2 mice.
Mir151 heterozygous N1F1 male mice were also backcrossed with C57BL/6JNarl
female mice for ten generations and performed speed congenic at N4 to obtained
Mir151 conventional knockout mice on a C57BL/6JNarl background. Genotyping of
N1F2 mice was performed by Southern blotting and by PCR. The PCR reaction mixture
contained 0.3- to 1-μg tail DNA, 200 μM dNTPs, 200 nM each of the primers, and 1 U
Taq DNA polymerase in 25 μL reaction buffers supplied by the manufacturer. The
primer sequences used for genotyping were shown in Table 1. ES cell gene targeting,
excision of neomycin resistance gene, blastocyst injection, and chimera production were
supported by the Transgenic Mouse Model Core Facility of the National Research
Program for Genomic Medicine, the National Science Council of Taiwan.
7.2.3. Animals
N1F2 male and female mice were used in all of the experiments in this study. N10F2
male mice (6~8 weeks) were only used in urethane induced model. All mice were
housed and bred in the Laboratory Animal Center of the Department of Bioscience
Technology of Chung Yuan Christian University (CYCU). The mice (n = 5/cage) were
maintained in a room with a constant temperature of 22 ± 1°C, relative humidity of 55 ±
10% and 12-h light/dark cycle, and fed standard rodent chow and purified distilled
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water ad libitum. Mice for long-term observation were kept and weighed every month
until the end of their life. The dead bodies of mice were collected, dissected, taken
pictures, and major organs including liver, heart, spleen, lung, kidney were preserved in
10% formalin. All animal experiments were approved by the Institutional Animal Care
and Use Committee of CYCU.
7.2.4. RNA preparation and reverse transcription
Tissues including lung, heart, brain, kidney, spleen, liver, thymus, and lymph node were
collected, washed with cold 1X PBS, and homogenized in T-PER buffer. Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions. For
mRNA qRT-PCR, 5μg total RNA was first reverse transcribed into cDNA by reverse
transcriptase using oligo dT and random hexamer as primers. For miRNA detection, the
TaqMan® miRNA RT kit and RT primers from Taqman miRNA assay were used
following the manufacturer’s instructions.
7.2.5. Quantitative real-time PCR
Expression of miR-151-3p and 5p were determined by TaqMan real-time PCR using
Taqman microRNA assays. All miRNA assays were run concurrently with a calibration
control, snoRNA-202, and were run in triplicate. Transcript of Ptk2, Epo, Phd3, Pgk,
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Vegf A and the reference gene Gapdh was determined by SYBR Green real-time PCR
and measured in two independent assays. The primer sequences used for qRT-PCR were
shown in Table 1.
7.2.6. Complete blood counts and differential counts
Whole blood was collected by puncture of the retro-orbital plexus of mice using
capillary tubes with anticoagulant. Complete blood counts (CBCs) and Differential
counts (DCs) was performed on EDTA•K2-anticoagulated blood using an automated counting device with the aid of Taiwan Mouse Clinc’s service.
7.2.7. Clinical chemistry
Whole blood was collected by puncture of the retro-orbital plexus of mice using
capillary tubes without an anticoagulant. Serum was diluted five times with distilled
water and serum blood chemistry was analyzed by an automated device. Parameters
analyzed included levels of total protein , albumin (ALB), globulin, total bilirubin
(BUN), aspartate aminotransferase (GOT), alanine aminotransferase (GPT), alkaline
phosphatase (ALP), amylase, lactate dehydrogenase (LDH), and γ
-glutamyltransferase, triglyceride (TG), total cholesterol (T-CHO), high density
lipoprotein-cholesterol (HDL-C), Na, K, Cl, Mg, P, Ca, Uric acid (UA), Fe, UIBC, and
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TIBC
7.2.8. Chronic hypoxia
N1F2 male mice of each genotype (18-20 weeks, weighing 33-54 g) were
separated into two groups. One group (hypoxic mice) was exposed to 10% O2 in a
well-ventilated, temperature-controlled hypoxia chamber for 6 hours. The other control
group (normoxic mice, control) which exposed to 10% O2 for 0 hour was maintained
under ambient normoxic conditions. At the end of exposure, mice were sacrificed, the
kidneys were quickly removed and then performed RNA extraction immediately or
rapidly frozen in liquid nitrogen. The frozen organs were stored at –80°C.
7.2.9. CoCl2 treatment
N1F2 male mice of each genotype (34-36 weeks, weighing 34-57 g) were
separated into two groups and injected i.p. with 60 mg/kg cobaltous chloride (CoCl2)
solution in 1X PBS or 1X PBS as control group. Animals were sacrificed 6 hours after
CoCl2 injection. The kidneys were quickly removed and performed RNA extraction
immediately or rapidly frozen in liquid nitrogen. The frozen organs were stored at –
80°C.
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7.2.10. Tumor analysis
Lung tissues were fixed in 10% formalin followed by 70% EtOH. Tumors were
counted and measured. The individual tumor size was measured. Tumors were graded
on a 4-stage grading system: macroscopic normal and on hematoxylin and eosin (H&E)
slides with typical adenomatous hyperplasia as grade I; nodule ≦ 2 (smaller than 1/4
lobe), normal part is still remained in the same lobe as grade II; nodule > 2 or larger
than 1/4 lobe, normal part is still remained in the same lobe as grade III; Completely
loss of normal tissue in the same lobe as grade IV.
7.2.11. Urethane-induced lung cancer model
The Mir151+/+, Mir151+/－, and Mir151－/－ mice (N1F2 n=7 for each group, N10F2
n = 12 for each genotype), matched by age (N1F2 23 weeks, N10F2 6–8 weeks old)
and weight (N1F2 31-47 g, N10F2 16–19 g) were used. These mice were injected i.p.
with 1 mg/g body weight urethane in normal saline once weekly for 6 (N1F2) or 4
(N10F2) consecutive weeks. At the end of the experiments, mice were sacrificed and
dissected their lungs. Lung tumor numbers and sizes were evaluated under a dissecting
microscope. Tumors were isolated from the normal part and quickly frozen in liquid
nitrogen for further analysis.
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7.2.12. Histological analysis
All tissues were fixed in 10% buffered formalin for 24 h, embedded in paraffin, cut
into 5–7-μm sections, and stored at 4oC in the dark. For histological analysis, the
sections were dewaxed before staining with hematoxylin and eosin (21). All kidney
sections were examined by Dr. SL Lin (Nephrology, Department of Medicine, National
Taiwan University Hospital), and lung sections were examined by Dr. WC Lin
(Department of Pathology, National Taiwan University Hospital)
7.2.13. Statistical analyses
Statistical analysis was performed by using ANOVA or Mann-Whitney test of Prism
software (GraphPad, California, USA). Data are expressed as mean with SDs. A value
of p<0.05 was considered significant.
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Chapter 8. Results
8.1. Generation and identification of Mir151 conventional knockout