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Recombinant DNA: Gene Manipulation, Transgenic Organisms, and Reverse Genetics, Lecture notes of Genetics

University of Sargodha (US)Genetics

The applications of recombinant DNA technology, focusing on gene manipulation techniques such as site-directed mutagenesis and in vitro mutagenesis, the creation of transgenic organisms including bacteria, yeast, plants, and mice, and reverse genetics methods like RNA interference and CRISPR gene editing. Topics include the use of reporter genes, transgenic organisms for research and agriculture, and ethical considerations.

Typology: Lecture notes

2019/2020

Uploaded on 01/14/2020

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Lectures 28 and 29 โ€“ applications of
recombinant DNA technology
I. Manipulate gene of interest
A. site-directed mutagenesis
B. in vitro mutagenesis by PCR
why?
C. reporter transgenes
- fuse regulatory sequences to reporter
- use lacZ (encodes ๏ข-galactosidase), gfp (jelly fish green fluorescent
protein) or luciferase (fire fly enzyme)
- eg: ceh-23::gfp
- why?
II. Transgenic organisms - introduce gene from one species into genome of
second species
A. transgenic bacteria โ€“ generally introduce gene on plasmid
- why?
1
pf3
pf4
pf5

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Lectures 28 and 29 โ€“ applications of recombinant DNA technology I. Manipulate gene of interest A. site-directed mutagenesis B. in vitro mutagenesis by PCR why? C. reporter transgenes

  • fuse regulatory sequences to reporter
  • use lacZ (encodes ๏ข-galactosidase), gfp (jelly fish green fluorescent protein) or luciferase (fire fly enzyme)
    • eg: ceh-23::gfp
  • why? II. Transgenic organisms - introduce gene from one species into genome of second species A. transgenic bacteria โ€“ generally introduce gene on plasmid
  • why?

B. transgenic yeast

  1. how?
  2. uses of yeast transformation C. transgenic plants
  3. can be transformed by particle gun or by Agrobacterium Ti plasmid
  4. Ti plasmid:
  5. engineering plants
  6. types of engineered plants a. luciferase (from firefly) b. b-galactosidase (from E. coli ) c. glyphosate resistant (from Salmonella ) d. Bt toxin (from Bacillus thuringensis ) Genetically modified plants

b. somatic โ€“ try to provide wild-type gene in some somatic cells

  • provide wild-type gene to some cells, restores production of protein in specific cells
  1. approaches to somatic therapy โ€“ use different vectors a. retrovirus โ€“ inserts into chromosome at random b. adenovirus โ€“ persists extrachromosomally c. adeno-related virus โ€“ advantages of adenovirus d. lentivirus โ€“ less prone to insertional mutagenesis F. an aside - cloning whole organisms
  2. still technically challenging
  • most embryos fail/die early
  • many of those that develop have defects
  1. can you clone yourself? G. another aside - stem cells
  2. what are they? a. share two characteristics i. unspecialized cells, renew through long periods through division ii. can be induced to differentiate
  3. three types a. embryonic i. ii. b. adult i. ii. produce type of tissue in which they reside 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
  4. potential benefits trophoblast- outer layer of cells, generally notpartof embryo blastocoel - cavity inner cell mass - becomes embryo, embryonic stemcells derivedfromthis

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

  1. restrictions on research a. privately funded, no restrictions b. publicly funded, can use but not derive embryonic stem cells for more info, see: http://www.nih.gov/news/stemcell/primer.htm IV. Reverse genetics A. what is it?
  2. forward genetics
  3. reverse genetics B. how do you get the mutation?
  • use knock-out techniques C. yeast knock-out mutations

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: E. mouse knock-out
  5. similar to procedure for generating transgenic mice