Lectures 28 and 29 applications of recombinant technology I. Manipulate gene of interest

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1 Lectures 28 and 29 applications of recombinant technology I. Manipulate gene of interest C A. site-directed mutagenesis A C A T A DNA B. in vitro mutagenesis by PCR T A 1. anneal primer 1 C A 1. fill in with DNA polymerase, seal nick with ligase 2. after replication in cell and C G C A C G why? 2. first cycle of PCR 3. after many rounds of PCR C. reporter transgenes - fuse regulatory sequences to reporter - use lacz (encodes b -galactosidase), gfp (jelly fish green fluorescent protein) or luciferase (fire fly enzyme) - eg: ceh-23::gfp ( Pceh-23 ) ceh-23 Pgfp ( gfp ) Head Tail Pceh-23 - why? gfp II. Transgenic organisms - introduce gene from one species into genome of second species A. transgenic bacteria generally introduce gene on plasmid h gh hgh - why? recombinan t HGH not for use by athletes recombinan t HGH recombinan t not for use HGH by athletes not for use by athletes recombinan t HGH not for use by athletes 1

2 B. transgenic yeast 1. how? 2. uses of yeast transformation C. transgenic plants 1. can be transformed by particle gun or by Agrobacterium Ti plasmid 2. Ti plasmid: normal Agrobacterium infection gall transfer ori 3. engineering plants Agrobacterium with engineered Ti plasmid transgenic plant 4. types of engineered plants a. luciferase (from firefly) leaf "punches" regenerate plantlets b. b-galactosidase (from E. coli) c. glyphosate resistant (from Salmonella) d. Bt toxin (from Bacillus thuringensis) Genetically modified plants infected cell growing tissue Crop Modification US World soybean herbicide resistant 93% 77% field corn herbicide resistant, Bt 86% 26% cotton Bt 93% 49% 5. GM (= genetically modified) plants a. many plants engineered to improve yield, etc. 2

3 b. potential benefits - improved yield - improved crops - reduced herbicide and pesticide use c. concerns D. transgenic mice why? reporters, models for human genetic disorders, etc. 1. DNA into embryonic stem cells 2. vector usually modified retrovirus ES cells from black mouse genex DNA neo R embryo from white mouse implant pseudopregnant mouse chimeric mice X selection (neomycin) E. transgenic humans gene therapy 1. can recombinant DNA technology be used to correct genetic disorders? 2. two general types a. germ line therapy not done, problems: b. somatic try to provide wild-type gene in some somatic cells - provide wild-type gene to some cells, restores production of protein in specific cells 3. approaches to somatic therapy use different vectors a. retrovirus inserts into chromosome at random b. adenovirus persists extrachromosomally c. adeno-related virus advantages of adenovirus d. lentivirus less prone to insertional mutagenesis 3

4 F. an aside - cloning whole organisms 1. still technically challenging - most embryos fail/die early - many of those that develop have defects 2. can you clone yourself? remove nucleus fuse cell produce cloned animal G. another aside - stem cells 1. what are they? a. share two characteristics i. unspecialized cells, renew through long periods through division ii. can be induced to differentiate 2. three types a. embryonic i. ii. b. adult i. ii. produce type of tissue in which they reside trophoblast - outer layer of cells, generally not part of embryo blastocoel - cavity inner cell mass - becomes embryo, embryonic stem cells derived from this c. induced pluripotent stem cells (ips) i. introducing 4 genes into differentiated cells can cause them to revert to a stem-like state ii. may cause elevated risk of producing tumors; still being investigated 3. potential benefits a. possibly used to treat number of different diseases egs: Parkinsons, diabetes, heart disease, spinal cord injury, duchenne muscular dystrophy, Huntington's, amyotrophic lateral sclerosis, multiple sclerosis, etc. embryonic adult ips 4. restrictions on research a. privately funded, no restrictions b. publicly funded, can use but not derive embryonic stem cells for more info, see: 4

5 IV. Reverse genetics A. what is it? 1. forward genetics 2. reverse genetics B. how do you get the mutation? - use knock-out techniques C. yeast knock-out mutations 1. ori ampr YIp D. C. elegans (also flies, mammals, plants, etc.) 1. RNA mediated interference (RNAi) 2. prepare double-stranded RNA from gene of interest 3. inject germ cells or early embryo 4. what it does: yfg uraura ura- yfg ura3 yfg select for ura select for urayfg E. mouse knock-out 1. similar to procedure for generating transgenic mice ES cells from black mouse DNA clone chromosome homology to gene X neor gene X tk X selection (neomycin and ganciclovir) implant pseudopregnant mouse embryo from white mouse neor gene X inactive chimeric mice F. CRISPR (= Clustered Regularly Interspaced Short Palindromic Repeats): A different way to perform gene therapy or knock out gene 1. Bacterial defense mechanism, produces a nuclease that clips DNA complementary to a guide RNA 2. Possible uses: 5

6 3. How CRISPR/Cas9 works A bacterial nuclease (Cas9) can be guided to any DNA sequence using a complementary RNA sequence (guide RNA) The nuclease creates a doublestrand break (DSB) that can be repaired by: Non-homologye end joining to disrupt a gene or Homologous recombination (HR) to insert a new/corrected gene 6