Natural DNA recombination

common method of DNA repair in bacteria and eukaryotes

in eukaryotes, also occurs in meiosis (chromosomal crossover)

restriction endonuclease cuts (at specific sequences) maternal and paternal DNA, ligase joins

exonuclease

cuts from the end of a DNA strand, releasing single nucleotides

endonuclease

cuts within a DNA strand, producing single-stranded nicks

create "sticky ends," free base pairs find equivalent DNA fragment and ligate

Recombinant DNA (rDNA) molecules

sequences generated from multiple sources

DNA sequences not found in nature

Taq1

bacterial restriction endonuclease used to create recombinant DNA

cuts, sticky ends/stabilization by H-bonds, ligation by DNA ligase

Recombinant proteins

Proteins that result from expression of rDNA

just because rDNA is incorporated into an organism doesn't guarantee its expression

expression vectors

DNA molecule into which recombinant DNA can be cloned

ex: bacterial plasmids are ideal expression vectors because they replicate as the bacterial cells replicate, so lots of recombinant DNA can be produced at once.

use of antibiotic-resistance gene in recombinant protein expression

along with target DNA sequence, an antibiotic-resistance gene is included in the plasmid

as a result, it's easy to separate those bacteria containing the desired plasmids (treat with antibiotic, only cells containing plasmid will survive)

Other vectors used in rDNA techniques

bacteriophages, retroviruses, bacterial or yeast artifical chromosomes

requirement of plasmid to be suitable for gene recombination

must contain at least one specific nucleotide sequence that's recognized by a restriction endonuclease

advantage of bacterial protein expression systems

easy to culture, grow fast, high yield of recombinant protein

drawback to bacterial protein expression systems

multi-domain eukaryotic proteins are often non-functional when produced in bacteria because conditions necessary for post-translational modification/molecular folding are not present

many proteins become insoluble and precipitate as inclusion bodies (because of post-translational glycosylation), and are therefore difficult to recover

Mammalian in vitro recombinant protein production

+ and -

+ produces functional proteins

-low yield, high cost, time-consuming

rH proteins in clinical use

recombinant human insulin

recombinant human growth hormone

recomb. blood clotting factor Vila. NovoSeven

recomb. Hep B vaccine (cannot be produced in vitro)

Nutropin, Humatrope, Genotropin, Norditropin, Saizen, Omnitrope

recombinant HGH

used to be obtained from pituitary glad of cadavers-->Creutzfeldt-Jakob disease

gene therapy vectors

functional gene inserted into the genome of target cells to replace disease-causing gene

uses viral vector, which 'infects' cell with genetic material

retroviruses

create DNA from their RNA genomes, integrate into chromosomes of host cells

gene therapy vector

adenoviruses

viruses that have double stranded DNA genomes and cause resp, intestinal and eye infections in humans (ex: common cold)

used in gene therapy

adeno-associated viruses

small, single stranded DNA viruses that can insert genetic material at specific site on chrom 19

gene therapy vectors

herpes simplex virus

double-stranded DNA viruses that infect neurons

gene therapy vector

non-viral options for gene delivery (gene therapy)

DNA can be directly introduced into target cells

drawbacks: can only be used in certain tissues, requires large amts of DNA

Why gene therapy has not become effective tx for genetic disease

1. short-lived nature--cell turnover

2. immune response

3. problems with viral vectors--toxicity, immune/inflammatory response, tissue targeting, virus's ability to recover ability to cause disease

4. multigene disorders

transgenic mouse

carries a foreign gene that has been deliberately inserted into genome

knockout mutation

replacement of gene segment by homologous recomination resulting in nonfunctional or 'null' allele

knock-in mutation

mutation is a point mutation that results in partially functional or non-functional allele

reporter constructs

promoter for a 'reported' gene that is linked to the inserted expression cassette and that can be assayed

visible marker (LacZ, GFP)

generation of transgenic mice

1. in vitro ESC recombination: recombination, gene marked, embryonic stem cells isolated from blastocysts (by electroporation), homologous recombination, cloning

2. in vivo breeding: ESCs expressing gene inserted into mouse blastocyst and implanted in pseudopregnant female mouse

3. babies=chimeras

4. chimeras crossed with wild type

5. heterozygous knockouts produced

6. bred to each other to create homozygous knockout

large mice

rat hormone gene--bigger than normal

used to study growth, development

oncomice

overexpressed oncogene--predisposition to cancer

Doogie mice

improved memory and learning capacity

enhanced fn at NMDA receptors, which are needed for brain to store new info

fat mice

prone to obesity due to carboxypeptidase E-deficiency

strong mice

disabled myostatin gene

cold-tolerant mice

lack a sodium channel that causes pain when exposed to cold