Final Flashcards
What is reverse genetics?
Approach to discover the function of a gene by analyzing the phenotypic effects of specific engineered gene sequences.
What is functional genomics?
Focuses on understanding how complex biological phenomenon emerge from the intricate, dynamic, and complex web of interacting DNA, proteins, and lipids found within the cell. Seeks to illuminate the genotype-phenotype relationship.
What is RNA interference?
A natural biological process in which small RNA molecules silence, or knockdown, expression of a specific target gene.
How does RNAi work?
Short interfering RNAs (siRNAs) targeting the gene of interest are synthesized and introduced into the cell-type of interest. The introduced RNA molecules are recognized as foreign and incorporated into the RISC complex. The dsRNA is then unwound, leaving the antisense strand. The couplex then uses the antisense strand to guide itself to complementary mRNAs that are subsequently destroyed by endonucleolytic cleavage.
In what was RNAi first discovered?
Petunias. Was originally called PTGS (Post Transcriptional Gene Silencing)
What is Unc-22?
Uncoordinated 22. Codes for non essential myofilament and is present in several thousand copies/cell.
How does RNAi work in mammalian cells?
It works post transcriptionally in two key steps. First dicer cuts the dsRNA up into 21-23nt fragments to produce siRNA. The antisense strand of the siRNA then guides cleavage, incorporating into the RISC complex and allowing the RISC to bind to the mRNA, causing recreation of that area.
Why does siRNA avoid PKR?
PKR is a kinase that is activated during infection but siRNA is not recognized by the PKR pathway so they do not induce the process.
What is the PKR pathway?
Found in mammeling cells, it causes a potent response to dsRNA. The PKR binds to the dsRNA, resulting in interference production and apoptosis. It also results in eiF2alpha leading to blockage of protein synthesis.
What is an alternative approach to RNAi mediated knockdown of gene expression?
(a) siRNA molecules 19-22bp in length are chemically synthesized and directly transfected into the target cell to achieve knockdown. This allowed unprecedented ability to perform reverse genetic experiments. (b) They also tried using shRNA expressing constructs and incorporating them into a variety of vectors and then directly transfected into the target cell, or packaged into viruses that infected target cells.
What are some practical aspects of RNAi?
Biological research: defining gene function (reverse genetics), defining biochemical pathways
Therapeutic treatment: cancer, viral infection, parasitic infection
Describe the general mechanism of RNAi.
A dsDNA is chopped up by dicer into a siRNA duplex. The siRNA binds to RISC, creating the siRNP and activating the RISC. The siRNA binds to mRNA and results in mRNA cleavage at the binding area.
Alternatively, you can use a hair pin precursor. The diver cuts up the precursor to make an miRNA which binds to the protein creating the miRNP. This binds to the mRNA and inhibits translation of the mRNA.
What can transient virus-based delivery of shRNA be used to do?
Can be used to vary the levels of RNA interference.
Compare knockdown and knockout strategies.
Knockdown: decreased expression, may be transient or stable knockdown
Knockout: gene deletion strategy. May be constitutive or conditional which may be cell type of tissue specific and/or controlled by inducible.
How can specific genes be studied genetically if there is no suitable mutant?
Create genetically modified organisms
What are the four general types of genetically modified organism?
Transgenic: expresses a transgene (trans)
Knockout: targeted disruption of an endogenous gene (cis)
Knockin: introducing a specific sequence alteration in a gene (cis)
Conditional knockout/knockin: modification occurs under a specific condition only (trans+cis approaches)
What are some different methods of inserting DNA into a cell for genetical modified organisms?
1) Microinjection of cloned gene into nucleus of newly fertilized egg
2) Transfection: incubate ES cells in solution that makes them take up the DNA
3) Electroporation: a high voltage pulse helps DNA into the cell
4) Retroviral vectors: a more natural was of getting genes into cells
Describe transfection
ES cells are incubated in solution that makes them take up DNA. But it is very inefficient as you need to identify cells that took up that DNA with a reporter such as drug resistance.
What is transgenics?
One approach to making genetically modified animals. Uses a pronuclear/oocyte injection of construct containing the DNA of interest. Injects DNA into the pronucleus of a mouse embryo which is then implanted into the mouse. Later give birth to transgenic mice.
What are some drawbacks of transgenic animals?
The DNA randomly integrates into the genome, potentially disrupting other genes. The eggs must be harvested and fertilized in vitro and more than one copy of the gene may get into the genome.
How are transgenes being used in human cells?
Transgenic fibroblasts can be used to generate induced pluripotent stem cells (iPS). These are not derived from human embryos, so there is no ethical concern as in embryonic stem cells and you can create lines that are genetically tailored to a patient.
Describe the knockout tactic of deleting a yeast gene.
Using homologous recombination, the open reading frame is deleted in vitro and replaced by a marker gene flanked on either side by sequences homologous to the deleted gene. Deletion can then be confirmed by southern blot or PCR analysis.
How are knockout mice created?
Using homologous recombination, the gene of interest in put into a vector. This vector is then mixed with embryonic stem cells from the embryo of donor parents. These cells are then allowed to culture and those that have the transgene are injected back into the embryo. Generally in first generation, will result in chimeric mouse (showing both traits).
Describe the Cre-loxP system.
System designed for conditional knockouts. It uses two parts: Cre recombinase (transgene), and LoxP DNA sequence (knockin). Are recombinase is a bacteriophage that mediates site specific recombination between two loxP sites.
What is floxing?
Floxing refers to the sandwiching of a DNA sequence between two lox P sites. This allows specific sequences to be deleted, translocated or inverted in Cre-lox recombination. Can also allow tissue specific gene expression.
How does Cre-Lox recombination function?
A Cre mouse is crossed with a floxed mouse with the target gene. Therefore, in the offspring, cells that are lacking the cre recombinase express the target gene, but in those that have active cre, the loxP sites are brought together, cutting out the target gene from functioning.
How can the Cre-Lox system be used to create tissue specific knockout?
By combining the Cre gene with a tissue-specific promoter, it will ensure that the cre protein is only expressed in certain cells, preventing knockout in the others.
How can the Cre-Lox system be used to create time specific knockouts?
Using tetracycline-regulated gene expression. By putting the tetO promoter in front of the cre and adding doxycycline later, you can activate the tetracycline and cause a knockout in adulthood, allowing the examinations of its effects on tissues. If you were to make the promoter in from of the rtTA gene (tetracycline activator protein) tissue specific, you could make a tissue+time specific knockout.
How do you do a somatic cell nuclear transfer? Use sheep example.
A donor egg is collected from a black face sheep and the nucleus is removed. The nucleus of a white faced sheep is then collected and fused to the enucleated cell with electricity. The fused egg is then allowed to form into an embryo and implanted into the black-faced sheep. A white faced lamb will be born (clone).
What are some problems with cloning?
Adult clone sudden death syndrome Weight problems Shorter telomeres Arthritis Lung cancer Low success rate
