Gene modulation techniques associated with cancer sciences

Transcript

1. RPG 599 Techniques & Analyses for Cancer Sciences (TACS) Gene Modulation Priyanka Rajan (Priyanka.Rajan@RoswellPark.org) Tao Dai (Tao.Dai@RoswellPark.org) 9/12/2022 1
2. Gene Modulation: Use of lab-based techniques to modify gene expression Production of therapeutic/nutritional molecules like Insulin Understanding the function of genes in normal or in disease Gene therapy to correct genetic disorders Drug/vaccine delivery https://answers.childrenshospital.org/gene-therapy-history/
3. Objectives Part 1: Select proper gene modulation based on the biological question • Knockout approaches CRISPR-Cas9 • Knockdown approaches (shRNA, siRNA) • Knock-in (Overexpression, Fluorescent, Luciferase) Part2: Select optimal approach to deliver the constructs into the cells • Chemical: Cationic lipid-mediated delivery • Biological: Viral delivery • Physical: Electroporation • An example of shRNA plasmid and tips of ordering 3
4. How to modulate gene expression? 4 RNA Interference (RNAi) = “Knockdown” Disrupt DNA sequence = “Knockout” Overexpress Transgene = “Knock-in” Enzymatic cutters 1. Knockout: Mutate DNA sequence to disrupt the expression of the gene by insertion or deletion of DNA base pairs 2. Knockdown: Silence gene expression at mRNA level by RNA interference methods without altering DNA sequence. 3. Knock-in : Insert gene into cells to produce in large amounts or transgene fused to reporters to study location and function of gene
5. Introduction of plasmid 5 Plasmid / Vector / Construct • Derived from bacteria • Circular piece of DNA capable of self-replication. • Easy to engineer • Produce large amounts of proteins • Produce enzymes, proteins that glow Covered in more detail in “Tools and Models of Molecular Oncology”, ICS lecture on – “Viruses and Cancer”. https://blog.addgene.org/plasmids-101-what-is-a-plasmid Activates transcription Cloning sites – insert - Transgene - RNAi - Enzymatic cutters Enable survival Select cells that contain the construct
6. Part 1. Gene modulation strategies Knockout by CRISPR- Cas9 6
7. Gene Knockout (KO) by CRISPR- Cas9 Cas9 – sgRNA complex recognizes target and generates double strand break Let’s watch a short video - Link 7 Target DNA Single guide RNA (sgRNA) complementary to target sequence, guides Cas9 to the target Cas9: nuclease enzyme that cuts both strands of DNA at target sequence • https://www.idtdna.com/pages/technology/crispr/crispr-genome-editing DNA damage repair Insertion/deletion of wrong nucleotides Insertion/deletion of correct nucleotides Mutation resulting in loss of expression No Mutation.
8. CRISPR-Cas9 example Western blot Flow cytometry Gurusamy D., et al. (2020). Cancer Cell, 37, 818-833. 8 3 guide RNAs per gene Negative control: Cells without sgRNA Positive control: Validation sgRNA Future TACS lectures: flow cytometry, western blot * Can be used with library screens *
9. Advantages and disadvantages of CRISPR-Cas9 based knockout Shared resource: Gene Targeting and Transgenic core facility Advantages Disadvantages Precise editing Each cell will have different mutation profile; need to optimize to generate >75% knockout Can be used to delete or insert genes Relatively expensive Stable or transient Constitutively active or inducible cas9 9
10. Knockdown Approaches RNA constructs mediate silencing of specific mRNA target ❑ Short-hairpin RNA (shRNA) ❑ Silencing RNA (siRNA) 10
11. Knockdown Approaches 11 https://www.gene-quantification.de/rnai.html Enzyme which cleaves double-stranded shRNA into 20–25 base pair fragments Catalyzes the degradation of mRNA complementary siRNA guide strand RNA-induced silencing Complex KD approach KD approach
12. Short-hairpin RNA (shRNA) • Commonly delivered by virus • Can be used for library screen 12 https://horizondiscovery.com/en/applications/rnai/shrna-applications Whichever depletion method utilized, you must confirm that the desired gene expression is reduced at mRNA and/or protein levels → qPCR → Western blot → Flow cytometry/ IF Akt2 knockdown by 3 shRNAs mRNA = RT-PCR Protein = western blot Weng Y., et al. Onc Letters. 13(3), 1553-1562 Introduce into cells Generate virus expressing the construct
13. Silencing RNA (siRNA) 13 • Synthesized in a laboratory • Commonly delivered via lipid based transfection • Short-term expression (~48hr) Ubby I.,et al. (2019). Oncogene, 38, 3415-3427. Testing efficacy of siRNAs si-1 and si-2 against p53 in two cell lines Negative Controls? : Positive control? : Validation? : No siRNA Si-scrambled (non complimentary to target sequence) Si-p53: siRNA that they know silences p53 Two cell lines, use shRNA/CRISPR/ inhibitors
14. Comparison of KD Approaches siRNA Pros • Faster KD than shRNA • Useful for studying essential genes shRNA Pros • Long-term, stable KD • Cheaper than siRNA • Useful for studying essential genes • Easily implanted into murine models 14 *Both can be used for library screens* Cons in vivo delivery is difficult Short-term KD More expensive than shRNA Cons Slower KD than siRNA Virus-mediated toxicity
15. Advances in siRNA based therapeutics • siRNA against Ephrin type-A receptor 2 EphA2 gene is being tested in patients with metastatic solid tumors - NCT01591356 • It is encapsulated in neutral DOPC liposomes with potential anti- neoplastic activity 15 https://www.cancer.gov/publications/dictionaries/cancer-drug/def/epha2-targeting-dopc-encapsulated-sirna
16. Knock-in approaches • Overexpression • Fluorescent reporters • Luciferase reporters 16
17. Overexpression • Switching ON of genes in cells • Plasmid constructs: Myc, KRAS • Constitutively active promoter • Commonly delivered by virus Adapted from https://www.creative-biogene.com/support/gene-overexpression-in-cell-lines.html Introduce into cells Generate virus expressing the construct Select and Verify mRNA and protein expression 17
18. 18 Fluorescent Reporters: express a fluorescent protein (FP) downstream gene of interest ex. GFP, RFP, YFP, TFP Reporter Knock-In: (Goh, Lim et al. PNAS. 2012) RalA and RalB genes fused to GFP Ghoroghi S. et al, elife, 2020
19. 19 Fluorescent Reporters: express a fluorescent protein (FP) downstream gene of interest ex. GFP, RFP, YFP, TFP Reporter Knock-In: Pros • Fluorescent proteins do not use substrates • Multiple fluorescent proteins can be used in a single model Cons • Fluorescent proteins limit fluorophore panel for flow • Fusing a reporter to a protein may alter protein folding, localization and function
20. 20 Luciferase Reporters: transgene linked to a luciferase reporter; expression measured using bioluminescence. Reporter Knock-In: Transgenic Models Pros • Luciferase reporters can measure fast biological processes • Luciferase reporters have high signal to noise ratio Cons • Luciferase reporters require a substrate • Fusing a reporter to a protein may alter protein folding, localization and function http://photobiology.info/Ohmiya.html
21. 21 shRNA (knockdown) siRNA (knockdown) CRISPR-Cas9 knockout (KO) GFP reporter knock-in (KI) Which method is which? a. Report location of gene b. Long-term depletion of a gene in vitro c. Short term depletion of a gene d. Disruption/deletion of DNA sequence of a gene
22. 22 shRNA (knockdown) siRNA (knockdown) CRISPR-Cas9 knockout (KO) GFP reporter knock-in (KI) Which method is which? a. Report location of gene b. Long-term depletion of a gene c. Short term depletion of a gene d. Disruption/deletion of DNA sequence of a gene
23. Part 2. Delivery strategy 23
24. How to deliver a construct into the cells? 24 Introduction of artificial nucleic acids into cells ➢ Physical - Electroporation ➢ Chemical – package construct in lipid based carriers, introduce into cells ➢ Biological – Package construct in artificial viruses. “Infect” cells with the virus Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector.
25. Deliver construct of interest into mammalian cells Physical approach: electroporation Chemical approach: lipid-based 25
26. Deliver construct of interest into mammalian cells Biological approach: viral vectors 26 Virus Pros Cons Retrovirus Stable expression Broad tropism Only infect dividing cells Moderate immunogenicity Insertional mutagenesis possible Lentivirus Stable expression Infects non-dividing cells Broad tropism Insertional mutagenesis possible Adenovirus Remains episomal High expression Infects non-dividing cells Large packaging capacity High transduction efficiency Broad tropism Transient gene expression High immunogenicity https://www.addgene.org/viral-vectors/
27. How to select optimal delivery strategies? Step 1: Based on the types and size of construct/plasmid • siRNA (small molecule) is easy to deliver with lipid-based delivery strategy • It is less efficient to deliver large plasmids than small plasmids • Plasmids need to be compatible with the delivery strategy. e.g., plasmids designed for lentiviral delivery should have virus-specific elements, such as LTRs. Step 2: Based on the experimental design: stable vs transient gene modulation • Stable: lentiviral/retroviral delivery, followed by antibiotics selection • Transient: adenoviral, lipid-based delivery Step 3: Based on the target cells: cell types and species (mouse or human) affect delivery efficiency • Generally viral based delivery has broad tropism 27
28. If protein A is found highly expressed in breast tumors and you would like to know whether protein A is important for tumor cell proliferation in vitro and tumor growth in vivo. Gene modulation strategy for cell proliferation assay (one-week experiment): • Construct: loss-of-function (siRNA, shRNA or CRISPR-Cas9 sgRNA) • Delivery: both transient & stable gene modulations are fine 1) Deliver siRNA using lipid-based approach 2) Deliver shRNA or CRISPR-Cas9 sgRNA using lentiviral system If the tumor cells are to be used in xenograft mouse study (one-month experiment), stable gene modulation is preferred. • Deliver shRNA or CRISPR-Cas9 sgRNA using lentiviral system into breast cancer cells, followed by antibiotics selection for 2 passages. These cells will now have stable protein A knock-down/out and are ready for xenograft mouse study. 28
29. How to obtain viral vectors for gene modulation? 1) Glycerol stock (bacteria) 2) Purified plasmid 3) Ready-to-use virus 29
30. An example of shRNA lentiviral plasmid Promoter driving shRNA expression Reporter gene Antibiotics resistance gene (mammalian cells) Lentiviral elements Antibiotics resistance gene (bacteria) Ori: Origin of replication Replication in bacteria Replication in HEK293T cells (express SV40 large-T antigen) 30
31. Some plasmid can be customized: Choose reporter gene Choose promoter Choose products (virus or glycerol stock) Choose targeted region (ORF vs 3’UTR) 31
32. 32 General protocol for lentiviral transduction 1.) On the day of transduction, remove existing media from the target cells which should be at ~70%-80% confluence in a 6 or 12 well dish. 2.) Apply the virus supernatant + 8ug/ml polybrene* directly on top of cells. The volume should completely cover the cells (ie. 1.0 ml for 6 well plate.) 4.) Return plate to incubator and incubate for 3-6 hours at 37°c / 5% CO2. 5.) Add at least 1 ml normal target cell media for a total volume of >2 ml. Incubate overnight at 37°c. 6.) The next day, change the media or split the cells if needed. 7.) After splitting the cells, supplement media with antibiotics (use the lowest concentration that kills all parental cells; performing antibiotics killing curve is highly recommended for each cell line) 8.) Replace media with antibiotics selection media every 3-4 days. Select for resistant colonies. Adapted from gene modulation share resource website. See below website for retroviral transduction protocol and retroviral/lentiviral production protocol https://www.roswellpark.org/shared-resources/gene-modulation-services# * Polybrene promotes infection by preventing electrostatic repulsions that can occur between the virus and the cell membrane.
33. 33 Trouble shooting--using lentiviral shRNA as an example: Problem Possible Cause Suggested Solution Few live cells after antibiotics selection or do not see good reporter fluorescent signals under microscopy Viral titer is low Use more virus, make/buy fresh virus and/or use smaller dish/plate for transduction Antibiotics concentration is too high Use lowest antibiotics concentration that kills 100% parental cells Do not see gene knock-down after antibiotics selection Do not wait enough time to check gene expression Wait 2-3 days before checking mRNA level Wait 3-4 days before checking protein level shRNA does not work Try other shRNAs
34. Applying NIH standards of experimental rigor & reproducibility Examples: • Controls: include non-targeted RNA, non-transfected cells, cell lines that already have the manipulation as a positive control. • One or more of the gene modulation strategies for validation. Eg- shRNA as a second approach to validate your CRISPR-Cas9 based KO. • Validate in multiple cell line models. • Usage of multiple sgRNAs, shRNA or siRNA sequences (n=2 minimum) 34 Rigor: The scientific methods for designing proposed research (controls, randomization, sample size) & performing such methods according to the conduct of research
35. Applying NIH standards of experimental rigor & reproducibility Examples: • Confirm genetic manipulation at DNA, mRNA, & protein levels, ensure it is cell/tissue-specific if specified • Validate all shRNA/siRNA constructs • Ensure that all your reagents are fully validated/not expired, from reputable vendors • Repeat experiments 2-3 times with adequate sample size • Record all procedures & methods of analyses 35 Reproducibility: Well-defined and validated experimental methods and analyses
36. https://www.roswellpark.org/shared-resources/gene-modulation-services# Individual shRNA in lentiviral/retroviral vectors Individual gene expression plasmid in lentiviral vector Contact details: Director: Dr. Irwin Gelman Irwin.gelman@roswellpark.org Renae Holtz Renae.holtz@roswellpark.org 36
37. Pyruvate kinase is a rate-limiting glycolytic enzyme that has two major isoforms M1 and M2. Research has shown that most cancer cells exclusively express M2 isoform. How can we study the advantages of M2 isoform over M1 isoform in tumor cell progression? Q1: How can we study the effects of PKM2 on tumor cell proliferation? • Knock-down using siRNA/shRNA? • knock-out using Cas9 and sgRNA? • What should be the control for each strategy? Christofk et al., Nature 2008 37 Q2: Can we replace PKM2 with PKM1 in tumor cells? • Knock-down/knock-out PKM2 and rescue with PKM1 • What should be the control? Q3: Can we compare the effects of PKM2 and PKM1 on tumor cell proliferation? • Knock-down/knock-out PKM2 and rescue with PKM1 or PKM2 • How many groups do we need to have? Q4: If we are comparing the effects of PKM2 and PKM1 on tumor growth in a xenograft mouse model, what gene modulation strategies can we use? • Stable gene modulation • Antibiotics selection; different antibiotics selection in knock- down plasmid and gene expression plasmid
38. Pyruvate kinase is a rate-limiting glycolytic enzyme that has two major isoforms M1 and M2. Research has shown that most cancer cells exclusively express M2 isoform. How can we study the advantages of M2 isoform over M1 isoform in tumor cell progression? Christofk et al., Nature 2008 Cells were infected with retrovirus containing the empty vector, pLHCX, or pLHCX with Flag-tagged mouse M1 (mM1) or mouse M2 (mM2). After 2 weeks selection in hygromycin, the cells were infected with lentivirus containing the pLKO vector with control shRNA (c) or shRNA that knocks down PKM2 expression (kd). The cells were then selected in puromycin for 1 week. Total cell extracts were immunoblotted with antibodies for pyruvate kinase (recognizes both M1 and M2), Flag and GAPDH. 38
39. Pyruvate kinase is a rate-limiting glycolytic enzyme that has two major isoforms M1 and M2. Research has shown that most cancer cells exclusively express M2 isoform. How can we study the advantages of M2 isoform over M1 isoform in tumor cell progression? 39 Christofk et al., Nature 2008
40. Useful links • Designing guide RNAs • http://crispor.tefor.net/ • https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=criGri1&g=crispr • RNAi video https://www.youtube.com/watch?v=9O3uxdDD1cA • To learn more about transfection/transduction and protocol: https://www.addgene.org • How to calculate viral titer: https://horizondiscovery.com/ja/resources/videos/calculating-viral-titer • Recommended viral titer for common lines: https://cdn.origene.com/assets/documents/lentiviral/recommended_lentivirus_moi_for_common_cell_lines.pdf • shRNA ordering: • Gene modulation shared resource • Mouse & human; lentiviral or retroviral plasmids; not cover every gene; may have only one shRNA available for one gene • Horizon discovery: https://horizondiscovery.com/en/gene-modulation/knockdown/shrna • pGIPZ-shRNA (human), pLKO.1-shRNA (human & mouse), Smartvector-shRNA (human & mouse, regular or inducible), pTRIPZ-shRNA (human, inducible); all lentiviral plasmids; click the link to see pros and cons for each shRNA plasmid • Sigma: https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/advanced-gene-editing/shrnas-for- individual-genes • pLKO-shRNA (human & mouse) • siRNA ordering: • Horizon discovery: https://horizondiscovery.com/en/gene-modulation/knockdown/sirna/products/sigenome-sirna-reagents • siGENOME siRNA (human & mouse) • Gene expression plasmid ordering: • Gene modulation shared resource (not cover every gene) • Origene: https://www.origene.com/products/cdna-clones/lentiviral-particles/lenti-orf 40