Chapter 22 - Applications of Genetic Engineering and Biotechnology

genetic engineering

the alteration of an organism’s genome usually using rDNA

genetically modified organisms

organisms that have manipulated DNA in their cells

biotechnology

using living organisms to create products or processes that help to improve life

recombinant protein

protein produced from recombinant DNA

biopharming

production of valuable proteins in genetically modified plants or animals

biopharmaceutical products to treat human disease

what are recombinant protein products used for

-human gene is cloned into a plasmid

-recombinant vector introduced into bacterial host

-large quantities of transformed bacteria are grown

-recombinant human protein is recovered and purified from bacterial extracts

recombinant protein production in bacteria process

recombinant insulin

first therapeutic protein produced by recombinant DNA technology

the pancreas of pigs and cows from slaughterhouses

where was insulin previously extracted from

insulin

protein hormone which regulates glucose metabolism

after translation

when is natural preproinsulin processed

preproinsulin N-terminal is cleaved off → proinsulin 3 disulfide bonds form and C-terminal cleaved off → mature insulin

insulin maturation process

-synthetic oligonucleotides encoding insulin A and B chains are inserted into separate vectors (adjacent to E.coli lacZ gene)

-when recombinant plasmids are transformed into E.coli host cells → lacZ gene and recombinant DNA A or B are transcribed and translated as a unit (fusion protein)

-fusion proteins extracted from host cells and purified

-insulin A or B chains are cleaved from B-gal by cyanogen bromide and then purified

-subunits are mixed together to spontaneously unite and form intact mature active insulin

explain process of recombinant insulin protein production

fusion protein

hybrid protein consisting of the amino acid sequence of B-gal attached to amino acid sequence for A or B insulin subunits

-bacterial cells often cannot process and modify eukaryotic proteins (can’t add necessary carb or phosphate groups to proteins)

-eukaryotic proteins in bacterial cells often do not fold properly in their 3-D structure (makes them inactive)

problems with bacteria as recombinant models

use eukaryotic hosts like yeast, transgenic farm animals, or cultured eukaryotic cells

how to overcomes issues with bacterial recombinant models

biofactories

living factories that could continuously produce the desired therapeutic protein that can be isolated in a non-invasive way

place the gene for antithrombin adjacent to a promoter for beta casein (protein in milk) → a single goat can produce much more than humans

how can human antithrombin anti-clotting protein be produced and extracted from milk from farm animals

to stimulate the immune system to produce antibodies against disease-causing organisms to confer resistance

what is the goal of vaccines

inactivated vaccines are prepared from killed samples of the infectious virus or bacteria while attenuated vaccines contain live viruses or bacteria that cause a mild form of the disease

inactivated vs attenuated vaccine

vaccine that consists of one or more surface proteins from the virus or bacteria instead of whole pathogen

what is a subunit vaccine

hepatitis B vaccine and human papillomavirus vaccine

2 examples of subunit vaccines

• it can be easily grown and can provide a constant source of recombinant protein

• typically much cheaper compared to bacteria, yeast, or mammalian cells

advantages of expressing recombinant proteins in plants

no not yet

are there any FDA approved drugs using plants for recombinant proteins?

expressing antibodies against the Ebola virus → mice used to create monoclonal antibodies, and then the genes were introduced into the plant

what have tobacco leaves been used for in ongoing clinical trials

they have large sized leaves and relatively high yield of recombinant proteins

why are transgenic tobacco plants commonly used for expressing recombinant proteins (compared to other plants)

the ability to transport and store the vaccine in any condition (refrigeration and sterilization facilities aren’t always available)

what is a huge barrier to vaccine effectiveness in general

vaccines that can be synthesized in edible food plants

in response to the barrier of transporting and storing vaccines, what are scientists trying to produce

hard to control dose → how big is the food, how many to consumer, will it pass through digestion unharmed, etc

what would be hard to control with vaccines synthesized in edible food plants

DNA encoding proteins from a particular pathogen are inserted into plasmid vectors, which are then directly injected into an individual or delivered by a virus

how do DNA vaccines work

pathogen proteins encoded by the delivered DNA would be produced and trigger an immune response that could provide protection should an immunized person be exposed to the pathogen in the future

what occurs after DNA vaccine is injected into individual

low production of recombinant protein resulting in low immune response

major limitation in trials with HIV vaccines

resulted in congenital birth defects prompted swift vaccine development and production → early trials worked well in mice and monkeys but have just begun in humans

what did the Zika virus outbreak lead to

selective breeding

the breeding of natural or mutagen induced mutations to produce a desired result

selective breeding

before genetic engineering, what did farmers use to manipulate the genetic makeup of plants and animals

insect resistance, herbicide resistance, or added nutritional characteristics

what does agricultural biotechnology work to produce in plants

• improve growth characteristics and yield

• increase nutritional value

• provide resistance against herbicides, pests, viruses, and drought

main reasons for generating transgenic crops

variety of rice that produces beta-carotene which is a precursor of vitamin A

what is golden rice

to study gene function or for production biopharmaceuticals

2 common reasons why transgenic animals are produced

mastitis

infection of mammary glands which block milk ducts from reducing milk output and contaminates milk with pathogenic microbes

transgenic cows are generated with the ability to produce the enzyme Lysostaphin which can cleave the cell wall of most common bacterial pathogen → allows them to produce a natural antibiotic that fights off S. aureus infection

how are transgenic cows being used to prevent mastitis

cow produces milk lacking B-galactoglobulin (whey protein) which is believed to be the primary cause of milk allergies

how does transgenic cow produce hypoallergenic milk

they produce a growth hormone that shortens the time to grow a mature salmon

what are transgenic salmon used for

gene drive

a particular gene being transmitted to a majority of an individual’s offspring (more than half)

• by cutting and pasting from one chromosome to another

• by removing a chromosome that determines the sex of the mosquito

use nucleases to modify mosquito genes and make gene drives in what 2 possible ways

males are modified and when they mate with females they pass along the lethal gene to produce short-lived offspring that die before they reach adulthood

what does the genetic modification of A. aegypti mosquitos do

prognosis

likelihood of developing disorder or predicted course of disease

diagnosis

identification of genetic cause of disease or condition

~60 condition required to be tested immediately after birth

what is newborn screening

testing before birth to detect disorders which may need earlier intervention

what are prenatal genetic tests

position of the fetus is determined by ultrasound and then a needle is inserted through the abdominal and uterine walls → remove some amniotic fluid which contains fetal cells for testing

what does amniocentesis prenatal testing involve

taking cells from fetal portion of placental wall for testing

what is chorionic villus sampling (CVS)

isolating circulating DNA from dead cells (cell-free DNA) because mother has her DNA and baby’s

what do non-invasive prenatal tests involve

it’s sequenced to analyze haplotypes to distinguish between maternal and fetal DNA

what is done with cell-free DNA taken from blood of pregnant mother

haplotype

DNA variations which are inherited but do not undergo recombination during gamete formation

typically no unless there is specific family history

is preemptive genetic testing covered by insurance

1 copy of a haplotype from the mother and 1 from the father

for any chromosome, the fetus inherits what in terms of haplotypes

• paternal haplotypes inherited to the fetus P2

• maternal haplotypes inherited to the fetus M2

• maternal haplotypes not inherited to the fetus M1

in terms of haplotypes what would the mother’s blood contain

maternal haplotypes inherited to the fetus

would maternal haplotypes inherited to the fetus or maternal haplotypes not inherited to the fetus be present in excess amounts

restriction fragment length polymorphism (RFLP) analysis

identifies variation in sequences by cutting DNA with restriction enzymes into different sized fragments