Cloning of tumor suppressor genes from Gene-trapped transformed astrocytes using Inverse- PCR
Please cite this article as follows: PCR Encyclopedia (2005) 1: 122726-00
Cloning of tumor suppressor genes from Gene-trapped transformed astrocytes using Inverse- PCR
PCR Encyclopedia (2005) 1: 122726-00
Cloning of tumor suppressor genes from Gene-trapped transformed astrocytes using Inverse-PCR
Deepak Kamnasaran, PhD
Arthur and Sonia Labatts brain tumor research center, Hospital for Sick Children, Toronto, Ontario, Canada.
1.1. Introduction
Astrocytes are glial support cells of the nervous system that have a multitude of roles, including, providing a framework for the development of neurons during embryogenesis, and metabolic supplements for the maintenance and function of neurons. Transformed astrocytes are called astrocytomas, which are pathologically part of all primary brain tumors responsible for the top 10 causes of cancer-related deaths in humans. Astrocytomas are the most common sub-type of gliomas, with four increasing astrocytoma grades as classified by the World Health Organization according to histological findings. Glioblastoma multiforme (GBM) is the most common malignant astrocytoma, which despite current therapy (surgery, radiation and chemotherapy), has a median survival of ~1 year. One of the reasons for treatment failures, especially in GBMs, is the lack of suitable molecular targets. The etiology of astrocytomas is polygenic and is supported by the finding of at least five familial syndromes, such as Turcot syndrome and Neurofibromatosis Types I and II, with patients developing astrocytomas (Prados, 2002). Secondly, Cytogenetic studies have demonstrated aberrations involving human chromosomes 3, 4, 5, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21 and 22 in astrocytomas (Prados, 2002). These chromosomes harbor one or more genes with an alerted gene dosage that can contribute to astrocytomas. Finally, mutations in several major genes such as MDM2, DCC, EGFR, NF1, PTEN, Rb, p16, p19 and p14ARF have been identified among astrocytoma surgical specimens (Prados, 2002). Genes involved in angiogenesis (VEGF, TIE2, PDGFR, ANG1, ANG2), autocrine stimulation (EGF, TNFa), transcription factors and other growth factors have aberrancies in astrocytomas and are therefore excellent candidates. Our current quest to identify tumor suppressor genes (TSGs) in astrocytomas is at the verge of discovery.
Insertional mutagenesis strategies are commonly used for random screens in search of genetic modifiers. For example, transposon-tagging using the Sleeping Beauty system or RAGE has identified possible TSGs or oncogenes respectively, in several cancers (Collier et al., 2005; Dupuy et al., 2005; Wang et al., 1999). Here, I describe a method to induce gliomas in rodents by using astrocytes that have been transformed by random mutagenesis with gene trapping. Gene trapping has worked successfully in several organisms such as Drosophila and murine cells (Lukacsovich and Yamamoto, 2001; Stanford et al., 1998). This procedure works best when astrocytes with a known genetic alteration that creates susceptibility to the formation of gliomas, is used for further mutagenesis by gene trapping. Gene trapping creates one or more genetic alterations to increase the potential for glioma formation. Gene trapping was initially pioneered by Gossler and colleagues on a mammalian system using murine ES cells (Gossler et al., 1989), and shortly afterwards to create several mutant mouse lineages (Friedrich and Soriano, 1991). There are many types of vectors used for gene trapping, including those for exon trapping and enhancer trapping. I describe the use of gene trapping using the Splice acceptor-type of vector (Figure 1).
Figure 1: Schematic of a standard Retroviral splice acceptor type Gene-trap vector.
Gene trapping with this type of vector results in the integration of genes that are usually expressed. Integration tends to be biased to the 5 end of a gene, that is, in the first or second intron. The typical features of a splice acceptor type of gene trap vector include a promoter-less reporter gene with or without an ATG translation start sequence, and a selection marker (Figure 1). At the 5 end of the gene trap cassette, is a Splice Acceptor (SA) sequence. This sequence is usually derived from the murine Engrailed 2, adenovirus late major transcript or human BCL-2 genes (Ishida and Leder, 1999). The ฿-galactosidase gene is the most common reporter gene because of the advantages of stability, non-toxicity and inexpensiveness. However the ฿-lactamase and the Jelly fish GFP genes can be used as alternative reporters (Silverman et al., 1998; Liu et al., 1999). The ฿-neo and hygromycin genes are commonly used selection markers (Natarajan and Boulter, 1995a). At the 3 end of the gene trap cassette is a Poly A tail sequence usually derived from the Simian murine leukemia virus 40 (Friedrich and Soriano, 1991).
The expression of the gene trapped endogenous gene creates a chimeric transcript comprising at the 5 end part of the endogenous gene transcript and a 3 end fused to the gene trap cassette transcript (Figure 2).
Figure 2: An outline of the mechanism of Retroviral mediated Gene-trapping of an endogenous gene.
The fusion transcript produces a truncated protein, and most likely a null allele, due to the Poly-A signal of the gene trap cassette (Figure 2). Integration into the 3 end of a gene can inactivate the endogenous gene, however the reporter and resistance markers can become non-functional due to protein misfolding. In general, only one allele of a gene is trapped. With this method, heterozygous tumor suppressor genes (TSGs) are identified. The identification of the trapped gene can be achieved by the use of Inverse-PCR (Natarajan and Boulter, 1995b) and/or 5 RACE (Voss et al., 1998). The success of using 5RACE is poor due to factors such as poor quality of the RNA template and low fidelity of the Reverse transcriptase when using the 5 regions of RNA molecules. Inverse-PCR (Figure 3) is a standard PCR reaction except the PCR primers are designed in inverse orientations to each other and the DNA template for amplification is a self-circularized molecule.
Figure 3: An outline of a typical Inverse-PCR reaction.
This method is more favorable because of the use of genomic DNA as a template, problems with secondary structure of the template are not evident, cost effectiveness and less time consuming.
I describe several protocols that describe how to 1) isolate and characterize astrocytes from the murine central nervous system, 2) produce retroviruses with a Gene-trap genome, 3) produce and screen a Gene-trapped astrocyte library for transformed astrocytes, and 4) use Inverse-PCR to clone heterozygous TSGs that contribute to the transformation of astrocytes. These procedures have been successfully used to identify a novel TSG for astrocytomas (Kamnasaran and Guha, 2005).
2. Materials and Reagents
2.1. Isolation of astrocytes
Astrocyte medium: [DMEM (Wisent)/streptomycin-penicillin-actinomycin (Wisent)/10% Horse serum (Wisent) /10% fetal bovine serum (Wisent)/ 12.5 ng/ml epidermal growth factor (Invitrogen)]
70% ethanol (Bioshop)
Halothane (Sigma)
Dissecting medium: [50 mls of 10X Hanks balanced salt solution (Wisent), 2.5 g of glucose (BioShop), 3.5 g of sucrose (BioShop), 0.175 g of sodium bicarbonate (BioShop) plus 450 mls of double distilled water]
Digestion medium: [900 mls of double distilled water, 100 mls of 10 X Eagles MEM (Invitrogen) plus 90 mls of (13.33 g of sodium bicarbonate (Bioshop) and 22.22 g of glucose (Bioshop) in 500 mls)]. Take 2.5 mls of this stock and add 1.5 mls of 0.25% Trypsin (Wisent)
Dissecting microscope (Leica)
12 cm pair of scissors (sterile) (Fisher Scientific)
Two 3C forceps per mouse (sterile) (Fisher Scientific)
100 mm petri dishes (Fisher Scientific)
100 mm primaria tissue culture plates (Becton Dickinson)
Screw cap tubes (50 ml) (Fisher Scientific)
2.2. Analysis of astrocytes
2-well slide chambers (Nalge-Nunc Labtek)
Astrocyte medium (See section 2.1 for the recipe)
0.05% Trypsin (Wisent)
PBS (Wisent)
10X Blocking reagent (Roche) [make 1% in PBS (wisent), 0.5% in PBS (Wisent)]
Tween-20 (Sigma)
PBST [500 mls of PBS (Wisent) plus 0.5 mls of Tween-20 (Sigma)]
GFAP anti rabbit primary antibody (AB cam)
FITC anti rabbit secondary antibody (AB cam)
Screw cap tubes (15 mls) (Fisher Scientific)
100 mm Petri dishes (Fisher Scientific)
Coverslip (Fisher Scientific)
DAPI mounting medium (VectaShield)
Hemocytometer (Fisher Scientific)
Fluorescent microscope (Leica)
2.3. Production of Gene trap Retrovirus
EcoPack-293 cells (Clontech)
EcoPack medium: [50 mls of Fetal bovine serum (heat inactivated) (Wisent), 5 mls of Penicillin-streptomycin-actinomycin (Wisent) plus 445 mls of DMEM medium (Wisent)]
Lipofectamine 2000 (Invitrogen)
Opti-MEM medium (Invitrogen)
Screw cap tubes (15 mls) (Fisher Scientific)
0.45 μM filters (Fisher Scientific)
5 ml syringes (Fisher Scientific)
Filter system: [remove the syringe plunger, attach the syringe to a 0.45 μM Filter. Place tip of the filter into a 15 ml screw cap collecting tube. Add the supernatant to the syringe holder and use the plunger to force the viral liquid suspension through the filter].
2.4. Titering of Gene-trap Retroviruses
NIH 3T3 cells (ATCC)
NIH 3T3 medium:[445 mls of DMEM medium (Wisent), 50 mls of Fetal bovine serum (heat inactivated) (Wisent), 5 mls of streptomycin-penicillin-actinomycin (Wisent)]
Polybrene (Sigma)
24-well collagen I coated plates (BD Biosciences)
Hemocytometer (Fisher Scientific)
2.5. In-vitro transformation assays: Soft agarose assays
Agarose (Bioshop)
150 mm tissue culture plates (BD biosciences)
water bath set at 41oC
Astrocyte medium (see section 2.1 for recipe)
Agarose coated tissue culture plates: [add 10 mls of 0.5% liquid agarose to a 150mm tissue culture plate, let sit for at least 20 minutes to dry in tissue culture incubator]
Hemocytometer (Fisher Scientific)
80 μM cell strainer meshes (Fisher Scientific) [cut into three cm2 pieces for use]
Screw cap tubes (15 mls) [Fisher Scientific]
Sterile glass Pasteur pipettes with rubber medicine bulb [Fisher Scientific]
2.6. Cloning of Gene-trapped alleles by Inverse-PCR
DNAzol kit (Invitrogen)
PCR purification kit (Qiagen)
T4 ligase plus buffer (New England Biolabs)
dNTP mix (Fermentas)
Platinum Taq HiFi polymerase kit (Invitrogen)
Agarose (Bioshop)
Gel extraction kit (Qiagen)
pCR-XL TOPO TA cloning kit (Invitrogen)
Kanamycin (Sigma)
Plasmid Miniprep purification kit (Qiagen)
3. Methods
3.1. Isolation of astrocytes
1. Anesthetize the mice with halothane
2. Gently wipe the head of the mice with 70% ethanol
3. Decapitate the head with a 12 cm pair of scissors and place in a 100 mm petri dish
4. Wash with dissecting medium to remove excess blood and maintain moisture
5. Cut the skin away from the skull from the midline of the base of the head towards the nose, then remove the skin with a 3C forceps
6. Scoop the brain out by sliding the 3C forceps under the brainstem
7. Place the brain in a clean 100 mm petri dish
8. Position the brain ventral side up under the dissecting microscope (7X magnification)
9. Using 3C forceps, peel away the menninges to unfold the hemispheres
10. Dissect out the hippocampus with another sterile 3C forceps
11. Transfer the hippocampus tissue to a 50 ml screw cap tube containing 15 mls of digestion medium
12. Incubate at 37oC for 30 minutes
13. Use the pipetting suction force of a p1000 pipetteman tip to dissociate the tissue
14. Centrifuge 1200 rpm for 5 minutes
15. Gently discard about 14 mls of digestion buffer
16. Add 19 mls of Astrocyte medium and resuspend the cells
17. Plate the cells on two 100 mm primaria tissue culture plates
18. Incubate at 37oC, 5% CO2 in a tissue culture incubator for 3 days
19. Gently discard 9 mls of Astrocyte medium and replace with another 9 mls of Astrocyte medium
20. Change the growth medium every 7 days until confluency
3.2. Analysis of astrocyte cultures
1. Plate about 5000 primary astrocyte cultures per well of 2-well chamber slides
2. Top up volume to 800 ml with Astrocyte medium
3. Incubate overnight at 37oC, 5% CO2
4. Aspirate Astrocyte medium
5. Remove chamber wells
6. Put the slide in a 100 mm Petri dish
7. Briefly rinse 2X with PBS
8. Fix with ice-cold methanol for 5 minutes then aspirate
9. Fix with ice-cold acetone for 5 minutes then aspirate
10. Wash 3X with PBS, 3 minutes each
11. Add 10 mls of 1% Blocking reagent and incubate at room temperature for 30 minutes
12. Wash 1X with PBST for 5 minutes, with gentle rocking
13. Add 10 mls of 0.5% Blocking reagent plus 1/3000 dilution of GFAP primary antibody
14. Incubate at room temperature for 1 to 2 hrs, with gentle rocking
15. Wash 2X with PBST for 10 minutes each, with gentle rocking
16. Add 10 mls of 0.5% Blocking reagent plus 1/1000 dilution of FITC-labeled secondary antibody
17. Incubate at room temperature for 1 to 2 hrs, with gentle rocking
18. Wash 3X with PBST for 10 minutes each, with moderate rocking
19. Aspirate excess PBST
20. Add DAPI mounting medium and a coverslip
21. Visualize with a fluorescence microscope to quantify the % of GFAP immuno-positive cells (Green) per total number of DAPI-stained nuclei (Blue). Use only those primary astrocyte cultures with > 90% abundance for GFAP immuno-positivity for Gene-trapping.
3.3. Production of Gene trap retrovirus
1. Grow EcoPack-293 cells on 100 mm Collagen I coated plates with 10 mls of EcoPack-293 medium until 90% confluency is achieved
2. Aspirate excess medium and replace with 10 mls of OptiMEM medium
3. Add to a 15 ml screw cap tube 1 ml of OptiMEM medium
4. Add 120 μl of Lipofectamine 2000 followed by 24 μg of engineered Retroviral Gene-trap vector DNA (such as illustrated in Figure 1)
5. Incubate for 20 minutes at room temperature
6. Add the contents to EcoPack-293 Cells
7. Incubate at 37oC, 5% CO2 in a tissue culture incubator for 72 hrs
8. Collect the medium and centrifuge at 800 rpm for 2 minutes
9. Filter the supernatant with a 45 μM filter system
10. Make aliquots of 0.5 mls and store at 80oC prior to use
3.4. Titering of Gene-trap Retroviruses
1. Grow up twenty thousand NIH 3T3 cells in each well of 24-well collagen I coated plates
2. Incubate with NIH 3T3 medium overnight at 37oC, 5% CO2 in a tissue culture incubator
3. Replace medium with NIH 3T3 medium plus 8 μg/ml of polybrene
4. Add into each well serial dilutions of the filtered retroviruses
5. Incubate overnight at 37oC, 5% CO2
6. Replace medium with fresh NIH 3T3 medium plus appropriate concentration of antibiotics to select for resistance as encoded by the Retroviral gene trap cassette
7. Incubate for 6 days, then change the medium with NIH 3T3 plus antibiotics every three days
8. Count the number of resistant cells per well and calculate the multiplicity of infection (MOI)
MOI = number of resistant cells divided by the highest dilution factor used to obtain resistant cells
3.5. Construction of Gene trapped library
1. Count the number of astrocytes
2. Calculate the number of astrocytes needed for an MOI of 1.5 to 5
3. On the day before transduction, plate the appropriate number of astrocytes on 150 mm primaria tissue culture plates (maximum of 20 million per plate) and grow in 12 mls of Astrocyte medium
4. Aspirate the medium
5. Add 15 mls of Astrocyte medium plus 8 μg/ml of polybrene
6. Add the appropriate volume of filtered Gene-trap retrovirus
7. Incubate overnight at 37oC, 5% CO2
8. After 24 hrs, decant the medium and replace with fresh Astrocyte medium plus antibiotics as resistance encoded by the Retroviral Gene-trap cassette
9. Replace the medium with antibiotics every 2 days for 6 days
3.6. In-vitro transformation assays: Soft agarose assays
1. Trypsinize the Gene-trapped astrocytes with 0.05% of Trypsin
2. Collect the astrocytes, centrifuge at 1400 rpm for 2 minutes, remove the supernatant and resuspend very well in 5 mls of Astrocyte medium
3. Filter the cells into single cell suspensions by pipetting through 80 μM cell strainer meshes and collect into a 15 ml screw cap tube
4. Count the number of transduced filtered cells with a hemocytometer and make an assessment to ensure the cells are in single cell suspensions. Otherwise, re-filter repeatedly through 80 μM cell strainer meshes until the cells are in single cell suspensions.
5. Add one hundred thousand cells to a 15 ml screw cap tube
6. Top up volume with Astrocyte medium to 3 mls
7. Add 7 mls of 0.5% agarose solution at 41oC
8. Gently invert the tube a few times to mix and pour the contents immediately into a 0.5% agarose coated 150 mm tissue culture plate
9. Spread the contents by swirling the plate
10. Let sit at room temperature for 30 to 45 minutes to dry
11. Add 20 mls of Astrocyte medium plus antibiotics (as resistance encoded by the Retroviral Gene-trap cassette)
12. Change the medium every 7 days
13. Grow in soft agarose for 14 to 21 days
14. Pick soft agar clones (as illustrated in Figure 4) with sterile glass pipettes with aid of a microscope.
Figure 4: shows an example of a transformed astrocyte colony in soft agarose.
3.7. Cloning of Gene-trapped alleles by Inverse-PCR
Inverse-PCR primers must first be designed from the Retroviral Gene-trap vector. The sequence (preferably full or partial is acceptable) for the vector interval must be known. Two excellent freeware online softwares that design Inverse-PCR primers are:
Primo Inverse 3.4 (Java program):
http://www.changbioscience.com/primo/primoinv.html
FastPCR ฉ 1998-2005 v.3.7 (standalone for Windows): http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm
From the list of possible designed primers, choose the pair with Tms between 60-65oC and %GC between 50-55%. Furthermore, the primers need to be within a 1-2 kb maximum distance from the 5 end of the Retroviral Gene-trap vector to allow efficient cloning and identification of the targeted gene. Use a frequent restriction enzyme cutter that is within 1 kb downstream of the Inverse-PCR primer binding sites. Use Figure 3 as a guide in the design of Inverse-PCR primers and restriction enzymes for use.
1. Extract the genomic DNA from the transformed astrocytes using the DNAzol kit and resuspend in a maximum volume of 400 μl lysis buffer pH 7.5.
2. Digest about 1-500 ng of genomic DNA for 7 hrs to overnight with a frequent restriction enzyme cutter that cuts downstream of the Inverse-PCR primer binding sites.
3. Clean up the digested DNA with the PCR purification kit and elute in 40 μl of double distilled water
4. Setup a self-ligation reaction with:
a. 20 μl of digested genomic DNA
b. 3 μl of 10X T4 ligase buffer
c. 3 μl of T4 ligase
d. 4 μl of double distilled water
e. incubate at 16oC for 4 hrs
5. Setup the Inverse-PCR reaction:
a. both Forward and Reverse Inverse-PCR primers (50 ng/ml ) 2 μl
b. 10X PCR buffer 2 μl
c. 50 mM MgSO4 2 μl
d. 2 mM dNTPs mix 2 μl
e. ligated digested template 2 μl
f. water 9.8 μl
g. Platinum HiFi Taq polymerase 0.2 μl
PCR cycle:
94oC 3 mins
94oC 30 sec
58oC 30 sec
68oC 4 mins (but vary up to 10 minutes, depending on results)
repeat for 45 to 50 cycles
6. Electrophorese on 1% agarose gel
7. Cut out the bands (usually the largest as priority) and purify the Inverse-PCR product(s) using the Gel extraction kit
8. Elute in 30 μl of double distilled water
9. Clone about 4 μl of the Inverse-PCR product(s) with the pCR-XL TOPO TA cloning kit
10. Randomly pick 4 colonies from each cloned Inverse-PCR products, and grow up in 50 μg/ml of kanamycin
11. Isolate the plasmids using the Plasmid Miniprep purification kit
12. Sequence the plasmids using the M13 Forward and Reverse primers
3.8. Bioinformatic analysis to discern the Gene-trapped allele(s)
1. Take the sequence obtained from the cloned Inverse-PCR product and perform homology searches against the Mus musculus database of genomic and cDNA sequences using standard BLAST searches. Ensure that the sequence used for the homology searches is unique and is not part of a gene family or conserved DNA sequence motif or repeat.
BLAST searches (http://www.ncbi.nlm.nih.gov/blast/)
Set the following parameters:
Database = NR
Select Organisms = Mus musculus
Filter = Low complexity
Select Expect = 0.0001
Number of Descriptions = 1000
Number of Alignments = 1000
2. Retrieve the genomic structure information of the gene demonstrating the highest identity (homology) to the partial Inverse-PCR product sequence, from the Ensembl database (http://www.ensembl.org).
3. Obtain the genomic sequence of the Gene-trapped gene from Ensembl and reassess the alignment of the Gene-trap cassette with the endogenous gene using the BLAST2 software.
BLAST2 (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi)
Set the parameter:
Expect = 0.0001
The following information will be discerned:
A) Orientation of integration within the gene sense (plus/plus alignments) or antisense (plus/minus alignments)
B) Position within the gene with the possibilities of integration within the 5 upstream, 3 downstream, within an intron or within an exon.
4.1. References
1. Collier LS, Carlson CM, Ravimohan S, Dupuy AJ, & Largaespada DA. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature. 14, 436:272-6 (2005).
2. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG & Jenkins NA. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 436:221-6 (2005).
3. Friedrich G & Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5:1513-23 (1991).
4. Gossler A, Joyner AL, Rossant J & Skarnes WC. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science. 244:463-5 (1989).
5. Ishida Y & Leder P. RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells. Nucleic Acids Res. 27:e35 (1999).
6. Kamnasaran, D & Guha, A. GATA-6 expression in the mouse and human central nervous system. Developmental Brain Research. 160(1):90-95 (2005).
7. Liu HS, Jan MS, Chou CK, Chen PH & Ke NJ. Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 260(3):712-7 (1999).
8. Lukacsovich T & Yamamoto D. Trap a gene and find out its function: toward functional genomics in Drosophila. J Neurogenet. 15(3-4):147-68 (2001).
9. Natarajan D & Boulter CA. A lacZ-hygromycin fusion gene and its use in a gene trap vector for marking embryonic stem cells. Nucleic Acids Res. 23(19):4003-4 (1995a).
10. Natarajan D & Boulter CA. Isolation of genomic sequences flanking a retroviral insertion site using a novel PCR-based method. Gene. 161(2):195-8 (1995b).
11. Prados M. Brain Cancer. Atlas of Clinical Oncology. Hamilton (ON): BC Decker Inc (2002).
12. Silverman L, Campbell R & Broach JR. New assay technologies for high-throughput screening. Curr Opin Chem Biol. 2(3):397-403 (1998).
13. Stanford WL, Caruana G, Vallis KA, Inamdar M, Hidaka M, Bautch VL & Bernstein A Expression trapping: identification of novel genes expressed in hematopoietic and endothelial lineages by gene trapping in ES cells. Blood. 92(12):4622-31 (1998).
14. Voss AK, Thomas T & Gruss P. Efficiency assessment of the gene trap approach. Dev Dyn. 212(2):171-80 (1998).
15. Wang A, Pierce A, Judson-Kremer K, Gaddis S, Aldaz CM, Johnson DG, MacLeod MC. Rapid analysis of gene expression (RAGE) facilitates universal expression profiling. Nucleic Acids Res. 27:4609-18 (1999).
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