BIMM 100 Lecture 8

BIMM 100 Lecture 8 - BIMM100 Lecture 8: Expression...

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Unformatted text preview: BIMM100 Lecture 8: Expression cloning and tricks for inac<va<ng genes Read: 204 ­212 Also read classic experiment (expressing foreign genes in mice) How else can we study our cloned genes? •  Make protein from the cDNA –  General strategies and considera<ons –  Poten<al problems •  What to make? •  How to make it? •  How to purify it? •  How to test it? Expressing a cloned cDNA in bacterial cells •  Classic inducible protein produc<on method –  In the presence of IPTG (a lactose analog), RNA polymerase transcribes the lacZ gene, which is translated into β ­galactosidase –  This is under the control of the lac promoter •  Different promoters/inducers can allow experimental control of expression Expressing a cloned cDNA Can be any gene! “Trick” the bacteria by puYng your GOI (gcsf here) under control of the lac promoter, instead of lacZ. Called subcloning. Adding IPTG now forces the cell to generate your GOI! Now you need to purify the protein. Hint? Add a “tag” to the protein… Success stories ­ recombinant technology to the rescue Product Medial purpose Restric<on endonucleases More cloning! CloYng factors (VIII & IX) Hemophilia A & B Erythropoie<n (epo) Anemia Insulin Diabetes (type I) Growth hormones Growth hormone deficiencies Interferons Leukemia, hepa<<s, MS DNAse Cys<c fibrosis Important products, and products that are commonly abused Not just abuse of drugs for physical gain, though… Off ­label use of recombinant factor VIIa (rFVIIa)! hdp:// sciencenow/2011/04/ hospitals ­engaging ­in ­ risky ­use ­.html?ref=hp Some possible issues •  •  •  •  •  Expression (can be toxic to host cells) Purifica<on (some<mes can be very difficult) Immunogenic Inappropriate uses Not fully func<onal –  May need processing, modifica<on, glycosyla<on, and special help folding not possible in bacteria Expression in animal cells •  Two op<ons: –  Transient –  Stable •  Animal cells do not stably replicate and segregate plasmids •  Choices –  Cell type –  Induc<on system •  Promoters •  Inducers –  Tags (or epitope if you use an an<body): a sequence that encodes a pep<de or protein not found in the host cell Expression in animal cells •  First steps: –  Clone gene into specialized vectors –  Introduce plasmid into cells via transfec<on (lipids, biolis<cs, electropora<on, etc.) •  Transient: quick, inexpensive, easy Not all cells get the plasmid! Expression in animal cells Resistance gene for G418 •  Stable: need selectable marker like G418 (an an<bio<c that blocks protein elonga<on in prokaryotes and eukaryotes) •  Does not stay as plasmid (like in bacteria ­ it becomes integrated into the cell’s genome. Thought to be done with host cell’s machinery. Integrates randomly! Also: know retroviral systems (figure 5 ­33) Yet another way to study your gene and protein Green= myeloid promoter driving GFP expression Ondrej Svoboda Use expression vectors to look at expression and localiza<on of proteins by fusing a fluorescent tag onto the protein of interest Can get the same kind of data as from an in situ hybridiza<on, only now with live cells Alterna<vely, you can add a specific epitope to the end of your protein and probe for it with an an<body More fluorescent tagging of proteins: the development of brain cancer wt +ras (an oncogene) Mar<n Distel The inac<va<on of genes •  BIG ques<ons! –  Is a specific gene essen<al for viability? –  What is the func<on of a gene within the cell? –  What role to genes play in the development and maintenance of the whole organism? How can we disrupt genes? •  The expression of a gene can be decreased (or eliminated) by… –  Genomic disrup<on of the gene of interest •  Yeast •  Mice –  Condi<onal KO –  Tissue specific KO –  Post transcrip<onal gene disrup<on •  RNAi •  morpholinos Yeast ­ a college student’s best friend In addi<on to these fine products, yeast is also an essen<al organism for gene<c manipula<on and studies High efficiency of homologous recombina<on Ability to propagate as haploid and diploid cells Strong research community ­ gene KO and GFP ­tagged collec<ons of organisms! Gene inac<va<on in yeast •  Gene inac<va<on in yeast (S. cerevisiae) 1.  Make disrup<on construct 2.  Introduce it into yeast cells 3.  Select for cells that integrated the disrup<on construct 4.  Study the cells with the deleted gene of interest Yeast gene<cs reminder Gene inac<va<on (in yeast) Step 1: building a disrup<on construct Make primers with sequence homologous to <20nt to the DNA sequence on either side of the GOI, as well as to kanMX kanMX confers resistance to G418 in yeast Construct with kanMX and homologous sequence ends Gene inac<va<on (in yeast) Heterozygous mutant Transform cells ­ homologous recombina<on will allow the integra<on of the disrup<on construct into the genome. KEY? The homologous sequence ends… Select for cells that have undergone recombina<on by adding G418 This is a great test to see if a gene is essen<al for viability! Gene inac<va<on (in yeast) You can also perform a Southern blot on these spores ­did the construct insert at the correct loca<on (i.e. KO the GOI)? Also, can perform PCR to see if the gene product is really knocked out. Remember, even if the gene is not essen<al, you then may be able to analyze it’s phenotype to learn something about it. Ex: Do the spores grow slower? Maybe this gene is important for growth control? From yeast to mice (and other mammals) Steps: 1.  Make a targe<ng construct 2.  Introduce it into murine cells 3.  Select for desired embryonic stem cells –  Select for cells that incorporate the targe<ng construct –  Select against the cells that incorporated the targe<ng construct by random integra<on 4.  Generate mice in which the gene of interest is disrupted in every cell (ubiquitous KO) 5.  Study the effect of the gene disrup<on on the development and maintenance of a whole organism (the mouse in this case). Making a targe<ng construct A B C Gene X genomic DNA Exons A neo C tk Gene x targe<ng construct Regions of homology Neomycin: posi<ve selec<on ­ confers resistance to G418 Thymidine kinase (tk): nega<ve selec<on ­ confers sensi<vity to ganciclovir. Cells normally have tk, but this gene is from the herpes virus, which inhibits DNA replica>on in ES cells. Introduc<on of targe<ng construct into murine ES cells A neo C tk Transfec<on, electropora<on, etc A B C A A B B C C Selec<on of cells that incorporate the targe<ng construct Homologous recombina<on: occurs at a very low frequency. That’s why you need to add a selectable marker! Selec<on against cells that incorporate the targe<ng construct by random integra<on Random inser<on: occurs at a very high frequency. That’s why you need to add a selectable marker to prevent this! Why do we not want random inser<ons? Posi<ve and nega<ve selec<on Now we have targeted disrup<on of a gene in ES cells. What’s next? •  For ease, we will choose a simple method to detect the chimeric animals that we will create. –  Chimera: an animal with <ssues derived from transplanted ES cells and host cells. •  Easiest method? Coat color! What do we have? •  Targeted embryonic cells from a brown mouse heterozygous for our gene x (x+/x ­). Since we started with ES cells from a brown mouse, and this coat color is a dominant allele, the ES cells are also (A/A) What do we need? •  An early embryo capable of incorpora<ng ES cells –  Choose a recipient that has a different marker allele (lets say black hair ­ the genotype of this mouse is x+x+ (because it is wildtype for gene x) and a/a (because it does not have the dominant brown fur color). –  Recep<ve pseudopregnant mouse to serve as a “surrogate mother” •  Done with hormones, or by ma<ng with a “fixed” male How is it done? •  Very carefully! Inject the ESCs into the blastocoel of the black mice with a small needle. Then, implant the blastocoels into the uterus of the “foster mother.” Introduc<on of mutant cells into mice Remember: A is brown coat a is black coat You can use any selectable marker ­ coat color is just so easy to visualize! Genera<on of mice with ubiquitous disrup<on of gene x Remember: you need to get integra<on in germ cells, or else these genes will not get passed on to future genera<ons! You are only interested in mice that are brown (i.e. are derived from the targeted ES cells) Genera<on of mice with ubiquitous disrup<on of gene x How can you screen for homozygotes? Now, you can look at phenotypes to find a func<on! Why would you want a condi<onal KO, versus a ubiquitous one? •  What if the KO mouse is inviable? •  What if a gene is important for development? •  What if you want to isolate the func<on of a gene in a par<cular <ssue? So, we need a way to modulate the expression of genes in different <ssues One example of an inducible system: Cre loxP •  A recombina<on system derived from bacteriophage P1 that also func<ons in mouse cells Introduced by non ­ homologous recombina<on loxP: site specific DNA recombina<on sites Cre: enzyme that catalyzes recombina<on between LoxP sites : <ssue specific promoter you use to direct Cre expression The nuts and bolts of the cre loxP system recombina<on FYI: some animals don’t undergo homologous recombina<on •  For example, it appears that zebrafish do not (although literature is conflic<ng on this subject) –  What to do? •  Zinc finger nucleases: create and introduce an enzyme that combines a gene ­specific DNA ­binding region and a nuclease ­ it can specifically target areas in the genome and “chop” them up. –  Problems? Difficult and expensive! Remember: you don’t need to control gene expression at the transcrip<onal level! •  You can use post ­transcrip<onal modula<on of genes! –  One example? RNAi (RNA interference) ­ using dsRNA to degrade a par<cular mRNA transcript. Why would cells have devised a way to destroy dsRNA? RNAi strategy 1.  dsRNA is recognized by cells 2.  dsRNA is processed by Dicer (an RNA endonuclease) found in all metazoans, but not simpler organisms such as yeast into 23nt ds segments called small inhibitory RNAs (siRNAs) 3.  siRNA associate with the RISC complex, and are separated into single strands 4.  These single stranded siRNAs recognize complementary mRNAs and base pair to them 5.  Aser basepairing, the RISC complex cleaves the mRNA/siRNA hybrid molecules Producing specific siRNAs Make the siRNA in vitro with two different constructs 23 nts Producing specific siRNAs Have the cells make dsRNA for you (with an RNA hairpin) Your construct is made of sense and an<sense sequence ­ that’s why it basepairs and makes a hairpin Detec<ng if RNAi worked: in situ hybridiza<on Worm embryo in situs for mex3 mRNA expression following RNAi treatment Complete abla<on of mRNA! Another posdranscrip<onal technique: morpholinos •  Can also block gene expression with morpholinos: –  oligos that block small (~25 base) regions of the base ­pairing surfaces of RNA •  Can design them against the start codon (no protein is translated) •  Can design them against the splice sites of mRNA (specific exons could be removed) –  Doesn’t KO genes, but knocks down their expression. When might you not want to completely knock out a gene, but just knock down it’s func<on? ...
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