Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.
Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.
The long noncoding RNA TERRA is transcribed from the subtelomeric region of chromosomes and its transcription proceeds towards the chromosome ends, terminating within the telomeric repeat tract1,2. For this reason, TERRA transcripts consist of subtelomeric-derived sequences at their 5' end and terminate with telomeric repeats (UUAGGG in vertebrates)3. Important roles have been proposed for TERRA, including heterochromatin formation at telomeres4,5, DNA replication6, promoting homologous recombination among chromosome ends7,8,9, regulating telomere structure10and telomere length homeostasis2,11,12,13. Furthermore, TERRA transcripts interact with numerous extratelomeric sites to regulate widespread gene expression in mouse embryonic stem (ES) cells14. In line with these evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at telomeres1,2,15. In addition, TERRA has been reported to form nuclear aggregates localizing at the X and Y chromosomes in mouse cells2,16. These findings indicate that TERRA transcripts undergo complex dynamics within the nucleus. Understanding the dynamics of TERRA molecules will help define their function and mechanisms of action.
The MS2-GFP system has been widely used to visualize RNA molecules in living cells from various organisms17,18. This system has been previously used to tag and visualize single-telomere TERRA molecules in S. cerevisiae12,19. Using this system, it was recently shown that yeast TERRA transcripts localize within the cytoplasm during the post-diauxic shift phase, suggesting that TERRA may exert extranuclear functions20. We have recently used the MS2-GFP system to study single-telomere TERRA transcripts in cancer cells21. To this aim, we employed the CRISPR/Cas9 genome editing tool to integrate MS2 sequences at a single telomere (telomere 15q, hereafter Tel15q) and obtained clones expressing MS2-tagged endogenous Tel15q TERRA (TERRA-MS2 clones). Co-expression of a GFP-fused MS2 RNA-binding protein (MS2-GFP) that recognizes and binds MS2 RNA sequences enables visualization of single-telomere TERRA transcripts in living cells21. The purpose of the protocol illustrated here is to describe in detail the steps required for the generation of TERRA-MS2 clones.
To generate TERRA-MS2 clones, a MS2 cassette is integrated within the subtelomeric region of telomere 15q, downstream of the TERRA promoter region and transcription start site. The MS2 cassette contains a neomycin resistance gene flanked by lox-p sites, and its integration at subtelomere 15q is performed using the CRISPR/Cas9 system22. After transfection of the MS2 cassette, single clones are selected and subtelomeric integration of the cassette is verified by PCR, DNA sequencing and Southern blot. Positive clones are infected with a Cre-expressing adenovirus in order to remove the selection marker in the cassette, leaving only MS2 sequences and a single lox-p site at the subtelomere 15q. Expression of MS2-tagged TERRA transcripts from Tel15q is verified by RT-qPCR. Finally, the MS2-GFP fusion protein is expressed in TERRA-MS2 clones via retroviral infection in order to visualize MS2-TERRA transcripts by fluorescence microscopy. TERRA transcripts can be readily detected by RNA-FISH and live-cell imaging using telomeric repeat-specific probes1,2,15,23. These approaches provide important information on the localization of the total population of TERRA molecules at single cell resolution. The generation of clones containing MS2 sequences at a single subtelomere will enable researchers to study the dynamics of single-telomere TERRA transcripts in living cells, which will help define the function and mechanisms of action of TERRA.
1 . Selection of Neomycin Resistant Clones
2. Screening of Neomycin Resistant Clones
3. Verification of TERRA-MS2 Transcript Expression by RT-qPCR
4. Production of a MS2-GFP Expressing Retrovirus
5. Visualization of TERRA-MS2 Transcripts in Living Cells
Figure 1 represents an overview of the experimental strategy. The main steps of the protocol and an indicative timeline for the generation of TERRA-MS2 clones in AGS cells are shown (Figure 1A). At day 1, multiple wells of a 6 well plate are transfected with the MS2 cassette and sgRNA/Cas9 expressing vectors (shown in Figure 1B). Two different subtelomere 15q-specific guide RNA sequences are cloned in the Cas9 nickase-expressing pX335 vector, generating two sgRNA-pX335 vectors that are co-transfected with the MS2 cassette. One well of the plate can be transfected with a GFP expressing vector to verify the transfection efficiency. At day 2, the cells are trypsinized and transferred from a single well to a 10 cm dish containing selective medium. At day 3, medium should be changed as most untransfected cells will be dead. Cells are grown in selection until clones are visible and ready to be picked. During this time, cells should not be trypsinized and culturing medium should be changed every 1-2 days for the first week and then every 2-3 days afterwards. Once single clones are visible, they are picked and transferred in a 96 well plate where they are allowed to grow until reaching 80-90% of confluence. At this point, clones are split in 4 different 96 well plates, which are marked in Figure 1 with a color code. Once the clones cultured in the DNA plate (green) reach 80% confluence, they are lysed and genomic DNA extracted for PCR screening; the clones in the backup plate (blue) will be kept in culture until the results of the PCR screening, while the red freezing plates are frozen at -80°C. Figure 2B shows representative results of a PCR screening of neomycin-resistant clones. For this screening, two sets of primers are used: one primer pair (MS2 primers) annealing within the neomycin gene and subtelomere 15q sequence is used to verify the presence of the MS2 cassette (a representative image of the primers localization is shown in Figure 2A). A second primer pair (CTR primers) annealing at an internal chromosomal region is used to verify the presence of false negative clones (primer sequences are indicated in Table 1). Negative clones for the integration of the cassette should be negative to the MS2 primers' amplification and positive to the CTR primers' amplification. Technical problems in genomic DNA extraction resulting in the absence of genomic DNA or its contamination would preclude PCR amplification from MS2 and CTR primer reactions. In these cases, the clones involved can be re-screened by PCR upon extraction of genomic DNA from the backup plate. Bands obtained from MS2 primers amplification of positive clones can be gel-extracted and sequenced using the MS2 primers, in order to confirm the presence of ten MS2 sequences.
The clones that are positive at PCR screening are cultured from the 96 well backup plate (the blue plate in Figure 1) to a 6 well plate, then lysed for genomic DNA extraction and Southern blot analyses. Figure 2D shows representative images of a gel (left) and membrane hybridized with a radioactively labelled MS2 sequence specific probe (right) of a Southern blot screening of PCR positive clones. For confirmation of the MS2 sequences integration at subtelomere 15q, genomic DNA extracted from each clone should be digested with two different restriction enzymes (NcoI and BamHI) in two separate digestion reactions. The NcoI and BamHI restriction enzymes are suitable for verification of the MS2 cassette integration at subtelomere 15q. Other enzymes can also be used. The gel shows a complete digestion of genomic DNA using the BamHI restriction enzyme, as indicated by the presence of a smear. The results from Southern blot indicate that one clone is positive for the integration of the MS2 cassette at subtelomere 15q while several clones show multiple integration events of the cassette, as indicated by the presence of multiple bands. A positive clone should be analyzed by a second Southern blot upon NcoI digestion of genomic DNA21. A representative image of a subtelomere 15q containing the MS2 cassette and the position of BamHI and NcoI restriction enzyme sites is shown in Figure 2C.
The clones positive to PCR and Southern blot analyses are infected with a Cre-GFP expressing adenovirus in order to remove the neomycin gene present in the MS2 cassette. Figure 3A depicts a MS2-tagged subtelomere 15q before and after Cre expression. An image of a clone positive for the MS2 cassette integration at subtelomere 15q infected with a Cre-GFP expressing adenovirus is shown in Figure 3B. For a complete removal of the neomycin gene in all cells, the infection efficiency should reach approximately 100%. As shown in Figure 3C, in order to verify the elimination of the neomycin gene, Cre-GFP infected cells are split in three plates after 48 hours from the infection. One plate will be cultured in the presence of neomycin. All cells in this plate should die within 6-7-days. In a second plate, cells will be allowed to grow in complete medium without selection up to 80-90% confluence. At this point, genomic DNA is extracted and analyzed by Southern blot. The third plate is cultured in complete medium to allow the preparation of frozen stocks of the clone and for RNA extraction and RT-qPCR analyses of TERRA-MS2 transcript expression. Figure 3D shows representative images of Southern blot analyses of a positive clone before and after Cre-GFP infection. DNA agarose gel (image on the left) confirms the complete digestion of the genomic DNA using the NcoI restriction enzyme. Southern blot analysis was performed using a radioactively-labelled MS2-sequence specific probe (image on the right). This analysis confirms the removal of the neomycin gene. The presence of a smear in the Cre-GFP infected sample confirms the telomeric integration of the MS2 sequences. The position of the NcoI restriction sites within the subtelomere 15q is shown in Figure 3A.
Once the elimination of the neomycin resistance gene is confirmed, the clones can be tested for the expression of TERRA-MS2 transcripts. To this aim, total RNA is extracted from each clone and run on a denaturating MOPS gel to confirm its integrity (Figure 4A). Ribosomal RNA bands should be visible at 4,700 bases (rRNA 28S) and 1,900 bases (rRNA 18S). Retrotranscription reaction is performed using a telomeric repeat-specific primer and reference gene-specific primer (Figure 4B, top) while qPCR analyses of TERRA expression are performed using two sets of primers: one primer pair annealing within the MS2 sequence and the subtelomere 15q sequence; a second pair of primers annealing within the subtelomere 15q. Primer sequences are indicated in Table 1. The graph presented in Figure 4B shows RT-qPCR analyses of TERRA expression from AGS WT cells and two different TERRA-MS2 clones. These analyses confirm the expression of TERRA-MS2 transcripts in the two clones at levels that are comparable to TERRA transcripts expressed from subtelomere 15q in WT cells. These data indicate that the two TERRA-MS2 clones selected are suitable for the analyses of TERRA-MS2 transcripts by live cell imaging.
In order to visualize TERRA-MS2 transcripts in living cells, the selected clones are infected with a retrovirus expressing the MS2-GFP fusion protein. Figure 5A shows the procedure to generate MS2-GFP expressing retrovirus, as described in protocol step 4. AGS WT cells and TERRA-MS2 clones are infected in glass-bottomed dishes. After 24 h from the infection, cells are analyzed by fluorescence microscopy. The specificity of the signal is confirmed by the presence of TERRA-MS2-GFP foci detected in the nucleus of TERRA-MS2 clones and not in AGS WT cells (Figure 5B). In a population of MS2-GFP expressing cells, heterogeneity in terms of MS2-GFP levels among cells is expected and cells expressing low levels should be chosen for the imaging analyses. Alternatively, the MS2-GFP expressing cells can be sorted by FACS prior microscopy analyses in order to collect the subpopulation of cells expressing low levels of GFP. We have previously detected TERRA-MS2-GFP foci in 40% to 60% of cells of AGS TERRA-MS2 clones21. In these cells, one to four TERRA-MS2-GFP foci were imaged per cell. TERRA-MS2-GFP foci showing distinct sizes and dynamics can be detected21. TERRA-MS2 clones can be used to study the dynamics of single-telomere TERRA transcripts in living cells. As an example of this application, Figure 5C shows representative images from microscopy analyses of a TERRA-MS2 clone expressing the MS2-GFP fusion protein and the telomere-binding protein TRF2 fused to mCherry in order to visualize MS2-tagged TERRA transcripts and telomeres in living cells. Using this approach, we have previously observed that TERRA-MS2-GFP foci co-localize with telomeres in 44% of the cells, indicating that TERRA transcripts only transiently co-localize with chromosome ends in AGS cells21.
Figure 1. Overview of the experimental strategy and indicative timeline for the generation of TERRA-MS2 clones in AGS cells. A) The steps described in the protocol and an indicative time line for the selection of TERRA-MS2 clones are shown. Cells are transfected in a 6 well plate with the linearized MS2 cassette and sgRNA/Cas9 expressing vectors. A color code is used to distinguish the DNA plate, the backup plate and the freezing plates indicated in the protocol section. B) The MS2 cassette consists of an 800 nt long subtelomere 15q sequence followed by 10 repetitions of the MS2 sequences and a neomycin resistance gene flanked by lox-p sites and terminates with a 300 nt long telomeric repeat tract at its 3' end. sgRNA/Cas9 expressing vectors were generated by cloning subtelomere 15q-specific guide RNA sequences in pX335 vector using the BbsI restriction site21,22. Two different sgRNA-pX335 vectors were generated and a 1:1 mix of the two vectors was used for the transfection. The sequences of the sgRNAs are indicated in Table 1. Please click here to view a larger version of this figure.
Figure 2. Representative images of PCR and Southern blot screening of neomycin resistant clones.A) Representative image of subtelomere 15q containing the MS2 cassette. The position of the MS2 primers (MS2-subtel15q-primer-S and MS2 primer AS) used for PCR screening is indicated. The loxP sites present within the cassette are shown in red. B) Representative image of PCR screening of neomycin resistant clones using two set of primers, MS2 primers and CTR primers (CTR prime S and CTR primer AS). C) Representative image of subtelomere 15q containing the MS2 cassette. BamHI and NcoI restriction sites and the MS2 probe used for the Southern blot screening are shown. D) Left: 10 µg of genomic DNA per clone were digested with BamHI and run on an agarose gel. The image was acquired after an overnight run at 30 volts. Right: genomic DNA digested with BamHI restriction enzyme was transferred to a positively charged nylon membrane and hybridized with a radioactively-labelled MS2 sequence-specific probe. The red box indicates the expected size of the positive band. The red arrow indicates one positive clone. Please click here to view a larger version of this figure.
Figure 3. Elimination of the neomycin resistance gene and validation experiments. A) Representative image of subtelomere 15q containing the MS2 cassette before and after Cre expression. The MS2 specific probe used for Southern blot analyses and NcoI restriction enzyme sites are shown. The loxP sites present within the cassette are shown in red. B) Fluorescence microscopy analysis of a TERRA-MS2 clone infected with Cre-GFP expressing adenovirus. Representative image acquired at a single focal plane is shown. MOI, multiplicity of infection, is the ratio between the number of viruses used for the infection and the number of host cells. Scale bar: 30 µm C) Overview of the experimental strategy used to verify the elimination of the neomycin gene. Cre-GFP infected cells are split in three plates after 48 hours from the infection for i) negative selection, ii) Southern blot analyses and iii) preparation of frozen cell stocks and RNA extraction. D) Southern blot analyses of a TERRA-MS2 clone before and after Cre-GFP adenovirus infection. 10 µg of genomic DNA were digested with the restriction enzyme NcoI. Agarose gel confirms that complete digestion is achieved in both clones (left). The digested genomic DNA was transferred to a positively charged nylon membrane and hybridized using a MS2 sequence-specific probe. Complete elimination of the neomycin gene is confirmed by the absence of the specific band. Please click here to view a larger version of this figure.
Figure 4. RT-qPCR analyses of Tel15q-TERRA and Tel15q-TERRA-MS2 transcripts expression. A) Representative image of a MOPS gel showing total RNA extracted from AGS WT cells and TERRA-MS2 clones. Bands corresponding to the ribosomal RNAs 28S and 18S are indicated. B) Top: A schematic of the MS2-tagged subtelomere 15q expressing TERRA transcripts. Primers used for TERRA retrotranscription (yellow) and qPCR analyses of Tel15q-TERRA (blue) and Tel15q-MS2 TERRA (red) are shown. The loxP site is shown in red. Bottom: RT-qPCR analyses of Tel15q-TERRA and Tel15q-MS2 TERRA transcripts expression in AGS WT cells and TERRA-MS2 clones. *p < 0.05, unpaired t-test. Please click here to view a larger version of this figure.
Figure 5. Live cell imaging analyses of TERRA-MS2 clones. A) An overview of the procedure to produce a MS2-GFP expressing retrovirus, as described in protocol step 4. A schematic of the MS2-tagged subtelomere 15q and TERRA transcripts recognized by the MS2-GFP fusion protein is shown on the right. B) Representative fluorescence microscopy image of AGS WT and two TERRA-MS2 clones expressing MS2-GFP. TERRA-MS2-GFP foci are indicated by arrows. Images were acquired using a spinning disc confocal microscope equipped with an EMCCD camera. The images were captured using a 100X/1.46 apochromat objective in an imaging chamber maintained at 37 °C with 5% CO2. A 488 laser was used as light source. Scale bar: 5 µm. C) Live cell imaging analyses of TERRA-MS2-GFP foci and TRF2-mCherry labeled telomeres. A co-localization event between a TERRA-MS2-GFP focus and a single telomere is shown. Images were acquired as in B, using 488 and 520 lasers as light source. A maximal projection of a z stack experiment performed at a single time point of time-lapse imaging experiment is shown. Scale bar: 5 µm. Please click here to view a larger version of this figure.
Primer name | Primer sequence | Application |
MS2-subtel15q-primer-S | TGCATTAAAGGGTCCAGTTG | MS2 primer forward used for PCR screening of neomycin resistant clones |
MS2 primer AS | CCTAACTGACACACATTCCACAGA | MS2 primer reverse used for PCR screening of neomycin resistant clones |
CTR primer S | TGT ACG CCA ACA CAG TGC TG | CTR primer forward used in PCR screening of neomycin resistant clones |
CTR primer AS | GCT GGA AGG TGG ACA GCG A | CTR primer reverse used in PCR screening of neomycin resistant clones |
Tel15q-S1 | GCAGCGAGATTCTCCCAAGC | Sense primer used to detect Tel15q and Tel15q-MS2 TERRA expression by qPCR |
hTel15q-AS | TAACCACATGAGCAATGTGGGTG | Antisense primer used to detect Tel15q and Tel15q-MS2 TERRA expression by qPCR |
Tel15q-MS2-AS | ATGTTTCTGCATCGAAGGCATTAGG | Antisense primer used to detect Tel15q-MS2 TERRA expression by qPCR |
TERRA-RT-primer | CCCTAACCCTAACCCTAACCCTAACCCTAA | Primer used for retrotranscription of TERRA transcripts |
sgRNA1 | TTGGGAGAATCTCGCTGGCC | Sequence of the short guide RNA 1 cloned in pX335 vector and used to direct Cas9 nickase enzymatic activity to subtelomere 15q |
sgRNA2 | TGCATTAAAGGGTCCAGTTG | Sequence of the short guide RNA 2 cloned in pX335 vector and used to direct Cas9 nickase enzymatic activity to subtelomere 15q |
Table 1: List of the primers used in this study. A list of the primers and the sequences of the sgRNAs used in this protocol are provided. Sequences are 5' to 3'.
In this article we present a method to generate human cancer cell clones containing MS2 sequences integrated within subtelomere 15q. Using these clones, the MS2-tagged TERRA molecules transcribed from the subtelomere 15q are detected by fluorescence microscopy by co-expression of a MS2-GFP fusion protein. This approach enables researchers to study the dynamics of TERRA expressed from a single telomere in living cells21. In this protocol, TERRA-MS2 clones are selected in the AGS cell line, which represents an interesting model system to study TERRA, since TERRA expression is upregulated in human stomach cancer samples24. However, the protocol described here can be adapted for the selection of TERRA-MS2 clones in other cell lines. The integration of the MS2 sequences can in principle be promoted at a subtelomere different from telomere 15q by using a specific MS2 cassette and CRISPR strategy. In the method presented here, a Cas9 nickase enzyme and double guide RNAs are employed in order to increase the specificity of integration22. Using this strategy, an overall 2% of positive clones are expected to be identified in AGS cells. Other versions of the Cas9 enzyme can be tested in the attempt to increase the success rate of the clones selection25. In addition, the following steps of the protocol are critical ones to take into account to maximize the efficiency of the clone selection:
I) In order to increase the chances of selecting the TERRA-MS2 clones, it is important to achieve a high transfection efficiency of the MS2 cassette and the CRISPR vectors. Poorly transfected cell lines will most likely require several rounds of transfection, clone selection and screening in order to select positive clones.
II) It is important to determine the precise concentration of the neomycin to use for the selection of the clones. Indeed, using a low concentration of the drug will result in false positive clones while a high concentration may preclude the identification of positive clones. The neomycin concentration indicated in this protocol is lethal to the AGS cells within 6-7 days from the addition to the culturing medium.
III) Clone picking is a critical step. Indeed, during this procedure it is required that only single clones are picked. For this reason, colonies that are too close to each other should be avoided in order to prevent mixing cells from different clones, resulting in the selection of a mixed population of clones which may contain MS2 sequences integrated at multiple genomic sites. These events should be identified during the Southern blot screening.
IV) The amount of genomic DNA extracted from a confluent 96 well DNA plate (protocol 2) is estimated to be around 5 µg and should suffice to screen every clone by PCR and Southern blotting with a single restriction enzyme digestion. However, in order to confirm the integration of the cassette at the expected telomere, it will be important to screen each clone by Southern blot by digesting the genomic DNA with two different restriction enzymes. To this aim, as indicated in the protocol, the clones positive at PCR screening should be cultured in 6 well plates. The amount of genomic DNA extracted from a well of a 6 well plate will be sufficient for at least two restriction digestions required for the Southern blot analyses.
In the protocol described here, the MS2 sequences are integrated within subtelomere 15q for a number of reasons: i) TERRA promoter region and TERRA transcription start sites have been identified on this subtelomere26,27; ii) TERRA expression from subtelomere 15q has been validated by using in vitro techniques4,27,28,29,30; iii) the subtelomeric region of the chromosome 15q has been sequenced and it contains a unique region adjacent to the telomeric repeat tract that can be targeted for the integration of the MS2-cassette21. PCR and Southern blot approaches were developed in order to confirm the single integration of the MS2 sequences within subtelomere 15q in the TERRA-MS2 clones. It would be interesting to also perform DNA-FISH experiments on chromosome spreads in order to visualize the chromosomal localization of the MS2 sequences in fixed metaphase chromosomes. However, the length of the 10xMS2 sequences (450 bp) imposes the use of very short probes as compared to the probes generally used in DNA-FISH experiments on chromosome spreads (Bacterial artificial chromosomes (Bac), cosmids or plasmids)31. This technical challenge has prevented us from visualizing the MS2 sequences on chromosome spreads which represents a limitation of the protocol. One further limitation of the method is the time and the effort required for selecting the TERRA-MS2 clones. In addition, specific laboratory set up and equipment for the use of viruses, radioactive material and microscopy analyses are required.
The method described here can be implemented for the generation of TERRA-MS2 clones in different cell lines in order to define the dynamics of TERRA in various biological contexts, such as during telomere dysfunction or cellular senescence, helping us to understand the function of TERRA in these processes. An interesting development of this approach will be the generation of TERRA-MS2 clones containing TetO repeats integrated at a specific subtelomere, including the MS2-tagged telomere. The expression of a TetR protein fused to a fluorescent protein (i.e., mCherry) will allow visualization of this particular telomere in living cells32. This approach will enable investigation of whether human TERRA transcripts localize and act in cis at the TERRA transcribing telomere and chromosome, or in trans by relocating to other chromosomes. This question in the biology of TERRA remains to be clarified.
The authors have nothing to disclose.
We are grateful to the staff of the advanced imaging facility of CIBIO at the University of Trento and the BioOptics Light Microscopy facility at the Max F. Perutz Laboratories (MFPL) in Vienna. The research leading to these results has received funding from the Mahlke-Obermann Stiftung and the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 609431 to EC. EC is supported by a Rita Levi Montalcini fellowship from the Italian Ministry of Education University and Research (MIUR).
AGS cells | – | – | Gift from Christian Baron (Université de Montréal). |
F12K Nut Mix 1X | GIBCO | 21127022 | Culturing medium for AGS cells |
L-Glutamine | CORNING | MT25005CI | Component of cell culturing medium |
Penicillin Streptomycin Solution | CORNING | 30-002-CI | Component of cell culturing medium |
Fetal Bovine Serum | Sigma Aldrich | F2442 | Component of cell culturing medium |
DMEM 1X | GIBCO | 21068028 | culturing medium for phoenix cell |
CaCl2 | Sigma Aldrich | C1016 | used in phoenix cell transfection |
HEPES | Sigma Aldrich | H3375 | used in phoenix cell transfection (HBS solution) |
KCl | Sigma Aldrich | P9333 | used in phoenix cell transfection (HBS solution) |
Dextrose | Sigma Aldrich | D9434 | used in phoenix cell transfection (HBS solution) |
NaCl | Sigma Aldrich | S7653 | used in phoenix cell transfection (HBS solution) and retrovirus precipitation |
Na2HPO4 | Sigma Aldrich | S3264 | used in phoenix cell transfection (HBS solution) |
TRYPSIN EDTA SOLUTION 1X | CORNING | 59430C | used in cell split |
DPBS 1X | GIBCO | 14190250 | Dulbecco's Phosphate Buffered Saline |
DMSO | Sigma Aldrich | D8418 | Component of cell freezing medium (80% FBB and 20% DMSO) |
G-418 Disulphate | Formedium | G4185 | selection drug for |
Gelatin solution Bioreagent | Sigma Aldrich | G1393 | cotaing of 96 well DNA plate and freezing plate |
Tris-base | Fisher BioReagents | 10376743 | Component of Cell lysis buffer for genomic DNA extraction |
EDTA | Sigma Aldrich | E6758 | Component of Cell lysis buffer for genomic DNA extraction |
SDS | Sigma Aldrich | 71729 | Component of Cell lysis buffer for genomic DNA extraction |
Proteinase K | Thermo Fisher | AM2546 | Component of Cell lysis buffer for genomic DNA extraction |
RNAse A | Thermo Fisher | 12091021 | RNA degradation during DNA extraction |
Agarose | Sigma Aldrich | A5304 | DNA gel preparation |
Atlas ClearSight | Bioatlas | BH40501 | Stain reagent used for detecting DNA and RNA samples in agarose gel |
ethanol | Fisher BioReagents | BP28184 | DNA precipitation |
Sodium Acetate | Sigma Aldrich | 71196 | Used for DNA precipitation at a 3M concentration pH5.2 |
Wizard SV Gel and PCR clean-Up system | Promega | A9282 | Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones |
Trizol | AMBION | 15596018 | Organic solvent used for RNA extraction |
Dnase I | THERMO SCIENTIFIC | 89836 | degradation of genomic DNA from RNA |
dNTPs mix | Invitrogen | 10297018 | used in RT and PCR reactions |
DTT | Invitrogen | 707265ML | used in RT reactions |
diethyl pyrocarbonate | Sigma Aldrich | D5758 | used to inactivate RNAses in water (1:1000 dilution) |
Ribolock | Thermo Fisher | EO0381 | RNase inhibitor |
MOPS | Sigma Aldrich | M9381 | preparation of RNA gel |
Paraformaldehyde | Electron Microscopy Sciences | 15710 | preparation of denaturating RNA gel (1% PFA in 1x MOPS) |
Superscript III Reverse transcriptase | Invitrogen | 18080-093 | Retrotranscription reaction |
Pfu DNA polymerase (recombinant) | Thermo Scientific | EP0501 | PCR reaction |
2X qPCRBIO SyGreen Mix Separate-ROX | PCR BIOSYSTEMS | PB 20.14 | qPCR reaction |
Cre-GFP adenovirus | https://medicine.uiowa.edu/vectorcore | 1174-HT | used to infect TERRA-MS2 clones in order to remove the neomycn gene |
Sodium Butyrate | Sigma Aldrich | B5887 | used to promote retrovirus particles production in phoenix cells |
PEG8000 | Sigma Aldrich | 89510 | Precipitation of retrovirus partcles |
35µ-Dish Glass Bottom | Ibidi | 81158 | used in live cell imaging analyses of TERRA-MS2 clones |