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Terms in this set (13)

recombinant DNA technology
1. produce DNA fragments using restriction enzymes that cut DNA at specific base sequences

2. link fragments to self-replicate forms of DNA, called vectors, to create recombinant DNA molecules

3. replicate these recombinant DNA molecules in a host organism to create thousands of exact copies of the DNA segment inserted into vector

4. retrieve cloned DNA insert in quantities large enough to allow further studies/modifications

DNA fragments cut using restriction enzymes -> link fragments to self-replicate forms of DNA (vectors) -> replicate the recombinant DNA molecules by putting them in a host organism -> retrieve cloned DNA for further studies

Cloning - production of identical copies of molecules, cells, or organisms from a single ancestor

Embryo splitting
1. egg fertilized outside body into 8-16 cell embryo

2. embryo divided by micromanipulation

3. individual cells implanted back into host for subsequent development

divide small egg cell into 2 -> put back in host

mimics how identical twins are produced

Nuclear transfer (cell fusion)
1. Eggs removed from animal, nucleus removed (enucleation)

2. enucleated eggs then injected w/pluripotent material nuclei taken from a 16-32 cell embryo w/desirable traits

3. cell fusion to introduce nuclei into egg cells

4. if transfer is successful, cells will begin to divide in vitro and produce a new embryo

5. if start w/16 cells, could end up w/16 embryos

6. When each cell develops to 16-32 cell stage, repeated for additional embryos

7. All embryos transferred to surrogate females for completion of development
Southern blot - used to separate DNA fragments using restriction enzymes for viewing

1. cleave DNA using restriction enzymes

2. DNA is loaded onto a gel made of agarose

3. DNA fragments separated by gel electrophoresis where an electric current is passed through the gel and since DNA molecules are negatively charged, migrate towards positive pole of electrical supply

4. as fragments move through gel, they separate by size, w/larger fragments only migrating short distances + small fragments migrating further

5. stain gel with DNA dye and expose to UV light and see a series of bands appear

2nd part of southern blotting
use a probe to identify fragment of interest

transfer fragments to a sheet of DNA-binding material

place membrane on top of gel, allowing a buffer solution to flow by capillary action through gel + membrane

movement of buffer transfers DNA fragments from gel to membrane

membrane exposed to a radioactively labeled probe (only those fragments attached to membrane w/complementary sequence will bind to probe)

a piece of x-ray film is placed over membrane + radioactivity exposes a part of film

1. DNA is cleaved
2. electrophoresis separates fragments by size
3. stain gel with DNA dye to see all the bands
4. place membrane on top of gel (cus you need to transfer)
5. buffer solution will flow by capillary action up
6. this transfers DNA from gel to membrane
7. radioactively labeled probe searches membrane for gene of interest (because of complementary sequence)
8. x-ray film placed over membrane to see gene of interest
Gene transfers for human disorders
change genetic make-up or influence genes directly

transfer normal genes into appropriate cells carrying defective copies

Retroviral vectors
retroviruses readily infect human cells
certain viral genes are removed, gene of interest is inserted

-> so it only inserts in ONE cell

insert vector into certain cells in the body and the vector DNA inserts into a human chromosome

gene then expressed (produces protein)

normal gene -> cloned into viral vector -> inject patient's white blood cells w/viral component -> inserts into human chromosome -> gene expressed (produces protein)

HUMAN GENE THERAPY
transfer a normal gene into cells to correct a specific disorder

human gene for enzyme ADA inserted into retrovirus and then transferred into white blood cells
(to treat genetic disorder called SCID)

normal ADA gene encodes enzyme to allow cells of immune system to mature properly -> no functional immune system without

1,500 clinical trials for gene therapy, still considered experimental

inefficient vector, can only insert retro vector into genes active in growth/division

difficult to direct where transferred gene goes, therefore transferred genes not expressed

improve -> fool immune system
direct genes to active regions
vectors that insert into any cell

FDA testing
preclinical -> test on animals
phase I -> human volunteers
phase II -> humans for toxicity/safety
phase III -> larger group

phase IV is approval phase

what would you target?
mitosis checkpoints -> gene to introduce proto-onco or tumor suppressor genes to balance out and control cell cycle
- treat leukemia and trisomy 21
Develop substitutes for damaged tissues and organs

millions of people lose function of tissues and organs and then must resort to mechanical devices

Scarcity of organs and organ donors is a serious problem

process ->
1. formation of new tissue or implant engineered functional cells to replace nonfunctional cells
2. isolate cells, change them and let them grow
3. synthetic polymer serves as a substrate for cells to help form correct tissues
4. biodegradable polymers break down over time or they cure non-degradable and remain in patient permanently
5. cells of nervous system, skin, muscles being tested

short form: 1. form new tissue, isolate cells, change them and let them grow
2. synthetic polymer acts as substrate for new tissue to have right structure
3. polymers break down over time in patient's body

bladder
1st bioengineered internal organ

liver cells
place hepatocytes in immunoprotective microcapsule that allows free movement of cell products, nutrients, oxygen

pancreatic cells
transplant pancreatic cells that produce insulin

skin cells
form new layers of artificial skin after severe burn

stem cells for larger organs
totipotent - form any type of cell in body
pluripotent - form most types of cells in body
multipotent - form few types of cells in body

embryionic stem cells
derived from inner cell mass in early embryos

somatic cell nuclear transfer
transfer nucleus from a somatic cell into an egg cell that was enucleated
grow this in culture and form ES cells

adult stem cells
found in umbilical cord, bone marrow, skin

spermatagonial cells - helps wounds heal, muscle