genetically engineered animals & gene therapy

transgenic animals

animals which have additional genes introduced

process of creating transgenic mice

take fertilised egg from organism - inject gene of interest before pronuclei fuse - screen offspring with PCR and breed positive mice to produce homozygotes

6 uses of transgenic animals

reporter genes to follow gene expression, models of disease, altered characteristics, pharming, insect control, general interest

how does chondromouse show collagen II production

green fluorescent protein inserted under control of the collagen II promoter - green fluorescence where collagen II is being made

eg of pharming

human antithrombin can be produced in goat milk

use of genetically engineered insects

reduced dengue fever transmission - engineered sterile males are released and mate with females - causes population crash

knockout gene def

wild type gene is replaced with an inactive gene to create a gene knockout

differences between transgenic and knockout mice

transgenic = random insertion and relatively high frequency. knockout = specific insertion, low frequency

how are knockout mice created

ESCs from blastocyst put on top of feeder cells - positive and negative selection of ESCs. black and white mice used - ESCs from black mice injected into white embryos - mate stripy offspring with white to produce all black knockouts

replacement knockout gene contains..

exons, neomycin resistance gene, thymidine kinase gene - marker

what kills cells with TK gene

ganciclovir

positive selection of recombinants

kills cells without transgene, treatment with neomycin

negative selection of recombinants

treatment with ganciclovir - kills cells with transgene but not in gene X

uses of knockout mice

test gene function - models of human disease eg CFTR

2 ways of treating diseases with genes

mutant gene replaced/supplemented by a normal one, therapeutic gene is introduced

targets for gene therapy

single gene disorders (CF, haemophilia, duchenne, sickle cell)

cancer, rheumatoid arthritis, erythropoietin

early attempts at CF gene therapy

added wild type CF gene to adenovirus vector - improvement for 8 weeks, then host immune system attacked vector and removed normal CF gene

adenovirus vector problems

common cold virus - we have antibodies, can elicit severe immune response, inserts randomly into genome

features of adeno-associated virus

very small, doesn't elicit immune response, infects dividing and non-dividing cells

process of stem cell gene therapy - for SCID

RNA of normal allele inserted into retrovirus - infects bone marrow cells removed from patient - viral DNA with normal allele inserts into chromosome, cells injected back into patient

CRISPR stands for

Clustered Regularly Interspaced Short Palindromic Repeats

how does cas9 nuclease work

makes double stranded cut within target region - guide RNA designed to match sequence in fault/abnormal gene