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Functional Genomics: The Knockout Approach

Functional Genomics: The Knockout Approach. Assessing physiological role of specific gene products. Eliminate given gene product and what happens? Gene silencing Insertional mutagenesis PCR Transposon T-DNA? Non-polar mutations (insert and recombine out). Gene silencing.

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Functional Genomics: The Knockout Approach

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  1. Functional Genomics: The Knockout Approach

  2. Assessing physiological role of specific gene products • Eliminate given gene product and what happens? • Gene silencing • Insertional mutagenesis • PCR • Transposon • T-DNA? • Non-polar mutations (insert and recombine out)

  3. Gene silencing • Normal everyday regulation: Transcriptional gene silencing (TGS) acts to prevent RNA synthesis through promoter methylation. While, post-transcriptional gene silencing (PTGS) acts to degrade existing RNA. • RNA interference – dsRNA causes degradation of corresponding mRNA leading to corresponding decrease in protein

  4. RNA interference (in detail) • Endogenously synthesized or exogenously applied dsRNA is processed by a ribonuclease III-like nuclease, Dicer, into 21- to 25-nt dsRNAs with 2- to 3-nt 3' overhangs. These dsRNA fragments, called short interfering RNAs (siRNAs), associate with an ~250- to 500-kD nuclease complex called RISC (RNA-induced silencing complex) and target this complex to complementary mRNAs. RISC then cleaves the targeted mRNAs opposite the complementary siRNA, which may render the mRNA susceptible to other RNA degradation pathways.

  5. Depends on your model • You can dip C. elegans and get effective gene silencing, but apparently there are issues in plants. • Some issues: • Different expression levels from same promoter • Never completely eliminates enzyme

  6. Insertional mutagenesis • Transposons, T-DNA (DNA transferred from A. tumefacians), or antibiotic cassettes • Forward genetics, select mutants or look for good coverage • No potential reversion, and generates an in vivo test tube to analyze protein function

  7. Bad points about knockouts • What phenotypes do you score?? • 1/3 of E. coli genes – unknown functions? • Pleiotropy – affecting pathways can throw a wrench in the works and cause multiple effects • Gene redundancy (70% of Arabidopsis genes are present in more than one copy)

  8. What is the probability? • If E. coli genes are ~ 1kb, how many insertions would we have to screen to have 99% confidence that we have insertions in every gene?? • Poisson distribution • http://info.bio.cmu.edu/Courses/03438/PBC97Poisson/PoissonPage.html

  9. Characterizing mutant • PCR • Southern • Marker segregation

  10. Establishing link between genotype and phenotype • Isolate independent mutations with same phenotype • Isolate revertants • Complementation

  11. Examples of functional genomics • Lipid metabolism and gluconeogenesis • Knockouts of malate synthase and isocitrate lyase (single genes) used to look at TAG role in seedlings • In the absence of glyoxylate cycle, TAG is still metabolized – respired • “Life finds a way…”

  12. Starch metabolism • Obtain knockouts in 7 of eleven genes • D-enzyme analysis reveals inadequacy of RNAi

  13. Interesting results • Knockout of plastidial phosphoenolpyruvate transporter results in compromised aromatic amino acid synthesis, underexpression of nuclear encoded photosynthetic proteins, and mesophyll-specific growth defects • This is one of many examples…

  14. Assessing phenotypes • Score defects in growth or gene expression • Peruse and write a brief review of 5 (that work) of the nine links in Table 1 of the C. elegans paper under the headings of gene function and gene expression… • http://www.dcb.org.tw/images/Lecture1.htm

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