39 Biotechnology Flashcards
FISH
a desired sequence is labeled with a fluorescent tag; the DNA is denatured and then allowed to hybridized with the chromosome; the flurescent tag appears wherever the sequence is found
what allows the visualization of the location of a desired DNA sequence on a spread of chromosome?
FISH
What allows us to see where a gene is expressed?
reporter gene
two examples of reporter genes
luciferase from fireflies and GFP from jellyfish
Benefit of reporter genes
since we can see where the gne is expressed it allows us to conduct studies on different regulatory sequences such as promoters and enhancers to see the effect of altering these sequences on gene expression based on monitoring the expression of the luciferase production
GFP
green flourescent protein - from jellyfish, will fluoresce under UV light - often joined with protein of interest allowing us to see where the protein is expressed throughout the organism
microarray analysis
allows comparision of the expression of a set of genes (compare mRNA) from different cell types
describe an example of microarray analysis
mRNA was isolated from cancerous andnoncancerous cells from women with breast cancer, the mRNA was converted to cDNA and labeled as red or green; it was incubated with the microarray chip whicha llowed it to bind to complementary sequences on the green chip; the computen analyzes which genes produce the mRNA in the cancerous cells compared to those produced by the noncancerous cells. Found overexpression or underexpression of some genes in the cancerous cells compared to noncancerous cells.
examples of gene products manufactured by bacteria and used as therapeutic proteins
human growth hormone, insulin, blood clotting factors
production of therapeutic proteins
coding sequences are put under a bacterial promotor and introduced into the bacteria via plasmid vectors
examples of gene products manufactured by bacteria and used for industrial applications
rennin for cheese, enzymes like bacterial proteases for detergents, meat tenderizers, amylases to degrade complex sugars
genetically modified crops are an example of?
transgenic plants
forward genetics
start with a mutant phenotype and seek out the gene that causes that phenotype; uses chromosome mapping to identify the gene
reverse genetics
start with a DNA sequence/genotype, alter its function or prevent its expression and observe the effects of the phenotype; frequently use transgenic organisms
knock-out mice
mice in which a gene has been turned off allowing the function of the gene to be assayed as we can look at the different in phenotype in the WT and knockout mice
knock down
altering the level of expression witout completely knocking out the gene; useful when completely knocking out the gene is not compatible with life - RNAi is used to decress the expression by decreasing the translation of or causing the degradation of mRNA
example of usage of knockdown mechanism
siRNA for ApoB gene in monkeys knocked down expression of ApoB reducing total level of cholesterol
crRNA
naturally occuring in bacteria to prevent invasion of bacteriophages - can be modified to alter gene sequences which has the potential to repair mutant sequences, add missing genes, or inactivate bad genes
gene therapy
introducing functional copies of a gene into individuals who have only defective copies of that gene
transgene
the introduced copy of the gene in gene therapy - in successful therapy will make the missing gene product and restore normal phenotype which may only occur in somatic cells
somatic gene therapy
treats, but does not cure the disease - all current gene therapies are of this type - nonheritable
germ-line gene therapy
would result in a heritable change and comes with moral and ethical considerations - do not currently have the ability to do this
What type of virus is used to introduce genes into a cell for gene therapy?
retroviral vectors
describe introduction of gene into cell for gene therapy
retroviral vector puts the transgene into the DNA of the host cell. After the transgene gets into a cell, it is transmitted to all progeny cells in the cell lineage by mitosis
potential problem with transgene
it may integrate in a way that disrupts the function of another gene
Other, non-retroviral, mechanisms for introducing a gene into a cell
direct injection, liquid capsules, nanoparticles, etc - can also deliver genome editing machinery like CRISPR
immune deficiency gene therapy trial
blood cells can be removed and targeted by retroviruses to deliver WT copy of the gene - being done in Severe Combined Immune Deficiency and Adenosine deaminase deficiency
hereditary blindness gene therapy trial
eye is accessible and partially protected from the immune system - a study with 9 patient showed improvement
cancer gene therapy trial
clinical trial in leukemia where patients immune cells were removed and modified to attack cancerous cells putting ~half into remission
