notes on genetic engineering

8/9/2019 Notes on Genetic Engineering

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Genetic Engineering

Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene

splicing are terms that apply to the direct manipulation of an organism's genes.!" Genetic engineeringis different from traditional breeding, #here the organism's genes are manipulated indirectly. Genetic

engineering uses the techni$ues of molecular cloning and transformation to alter the structure and

characteristics of genes directly. Genetic engineering techni$ues ha%e found some successes innumerous applications. &ome eamples are in impro%ing crop technology, the manufacture of synthetic

human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster o%ary

cells, and the production of ne# types of eperimental mice such as the oncomouse (cancer mouse) forresearch.

he term genetic engineering #as coined in *ac+ illiamson's science fiction no%el Dragon's -sland,

published in !!,0" one year before DNA's role in heredity #as confirmed in !0 by Alfred 1ersheyand Martha 2hase3" and t#o years before *ames atson and 4rancis 2ric+ sho#ed that DNA has a

double5heli structure.

he DNA5protein system is an ingeniously simple and etremely po#erful solution for creating all

+inds of biological properties and structures. *ust by %arying the se$uence of code #ords in the DNA,

innumerable %ariations of proteins #ith %ery disparate properties can be obtained, sufficient to generatethe enormous %ariety of biological life.

Engineering

here are a number of #ays through #hich genetic engineering is accomplished. 6ssentially, the

process has fi%e main steps.

5-solation of the genes of interest

5-nsertion of the genes into a transfer %ector

5ransfer of the %ector to the organism to be modified5ransformation of the cells of the organism

5&election of the genetically modified organism (GM7) from those that ha%e not been successfully

modified

-solation is achie%ed by identifying the gene of interest that the scientist #ishes to insert into the

organism, usually using eisting +no#ledge of the %arious functions of genes. DNA information can be

obtained from cDNA or gDNA libraries, and amplified using 829 techni$ues. -f necessary, i.e. forinsertion of eu+aryotic genomic DNA into pro+aryotes, further modification may be carried out such as

remo%al of introns or ligating pro+aryotic promoters.

-nsertion of a gene into a %ector such as a plasmid can be done once the gene of interest is isolated.

7ther %ectors can also be used, such as %iral %ectors, bacterial con:ugation, liposomes, or e%en direct

insertion using a gene gun. 9estriction en;ymes and ligases are of great use in this crucial step if it isbeing inserted into pro+aryotic or %iral %ectors. Daniel Nathans, erner Arber and 1amilton &mith

recei%ed the !


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endonucleases.

7nce the %ector is obtained, it can be used to transform the target organism. Depending on the %ector

used, it can be comple or simple. 4or eample, using ra# DNA #ith gene guns is a fairlystraightfor#ard process but #ith lo# success rates, #here the DNA is coated #ith molecules such as

gold and fired directly into a cell. 7ther more comple methods, such as bacterial transformation or

using %iruses as %ectors ha%e higher success rates.

After transformation, the GM7 can be selected from those that ha%e failed to ta+e up the %ector in

%arious #ays. 7ne method is screening #ith DNA probes that can stic+ to the gene of interest that #assupposed to ha%e been transplanted. Another is to pac+age genes conferring resistance to certain

chemicals such as antibiotics or herbicides into the %ector. his chemical is then applied ensuring that

only those cells that ha%e ta+en up the %ector #ill sur%i%e.

Applications

Molecular biologists ha%e disco%ered many en;ymes #hich change the structure of DNA in li%ing

organisms. &ome of these en;ymes can cut and :oin strands of DNA. >sing such en;ymes, scientistslearned to cut specific genes from DNA and to build customi;ed DNA using these genes. hey also

learned about %ectors, strands of DNA such as %iruses, #hich can infect a cell and insert themsel%esinto its DNA."

he first genetically engineered medicine #as synthetic human insulin, appro%ed by the >nited &tates4ood and Drug Administration in !=0. Another early application of genetic engineering #as to create

human gro#th hormone as replacement for a compound that #as pre%iously etracted from human

cada%ers. -n !=< the 4DA appro%ed the first genetically engineered %accine for humans, for hepatitis?. &ince these early uses of the technology in medicine, the use of GM has gradually epanded to

supply a number of other drugs and %accines.

7ne of the best5+no#n applications of genetic engineering is the creation of GM7s for food use(genetically modified foods)@ such foods resist insect pests, bacterial or fungal infection, resist

herbicides to impro%e yield, ha%e longer freshness than other#ise, or ha%e superior nutritional %alue.

-n materials science, a genetically modified %irus has been used to construct a more en%ironmentally

friendly lithium5ion battery."


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5Loss of function experiments, such as in a gene +noc+out eperiment, in #hich an organism is

engineered to lac+ the acti%ity of one or more genes. his allo#s the eperimenter to analy;e the

defects caused by this mutation, and can be considerably useful in unearthing the function of a gene. -tis used especially fre$uently in de%elopmental biology. A +noc+out eperiment in%ol%es the creation

and manipulation of a DNA construct in %itro, #hich, in a simple +noc+out, consists of a copy of the

desired gene, #hich has been slightly altered such as to cripple its function. he construct is then ta+enup by embryonic stem cells, #herein the engineered copy of the gene replaces the organism's o#n

gene. hese stem cells are in:ected into blastocysts, #hich are implanted into surrogate mothers.

Another method, useful in organisms such as Drosophila (fruitfly), is to induce mutations in a largepopulation and then screen the progeny for the desired mutation. A similar process can be used in both

plants and pro+aryotes.

5Gain of function experiments, the logical counterpart of +noc+outs. hese are sometimes performed

in con:unction #ith +noc+out eperiments to more finely establish the function of the desired gene. he

process is much the same as that in +noc+out engineering, ecept that the construct is designed to

increase the function of the gene, usually by pro%iding etra copies of the gene or inducing synthesis ofthe protein more fre$uently.

5Tracking experiments, #hich see+ to gain information about the locali;ation and interaction of thedesired protein. 7ne #ay to do this is to replace the #ild5type gene #ith a 'fusion' gene, #hich is a

:utaposition of the #ild5type gene #ith a reporting element such as Green 4luorescent 8rotein (G48)

that #ill allo# easy %isuali;ation of the products of the genetic modification. hile this is a usefultechni$ue, the manipulation can destroy the function of the gene, creating secondary effects and

possibly calling into $uestion the results of the eperiment. More sophisticated techni$ues are no# in

de%elopment that can trac+ protein products #ithout mitigating their function, such as the addition of

small se$uences that #ill ser%e as binding motifs to monoclonal antibodies.

5Expression studiesaim to disco%er #here and #hen specific proteins are produced. -n these

eperiments, the DNA se$uence before the DNA that codes for a protein, +no#n as a gene's promoter,is reintroduced into an organism #ith the protein coding region replaced by a reporter gene such as

G48 or an en;yme that cataly;es the production of a dye. hus the time and place #here a particular

protein is produced can be obser%ed. 6pression studies can be ta+en a step further by altering thepromoter to find #hich pieces are crucial for the proper epression of the gene and are actually bound

by transcription factor proteins@ this process is +no#n as promoter bashing.

Human genetic engineering

1uman genetic engineering can be used to treat genetic disease, but there is a difference bet#een

treating the disease in an indi%idual and changing the genome that gets passed do#n to that person'sdescendants (germ5line genetic engineering).="

1uman genetic engineering has the potential to change human beings' appearance, adaptability,intelligence, character, and beha%ior. -t may potentially be used in creating more dramatic changes in

humans.citation needed" here are many unresol%ed ethical issues and concerns surrounding this

technology, and it remains a contro%ersial topic.


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Human Genetic Engineering Background

9esearchers are currently trying to map out and assign genes to different body functions and

diseases.7nce the genes responsible for a disease are found, theoretically gene therapy should be able

to permanently cure the disease. -nteractions bet#een genes and gene regulators are comple and many

of these interactions are currently un+no#n.

History of Human Genetic Engineering

he first gene therapy trials on humans began in !B on patients #ith &e%ere 2ombined

-mmunodeficiency (&2-D). -n 0BBB, the first gene therapy success resulted in &2-D patients #ith a

functional immune system. hese trials #ere stopped #hen it #as disco%ered that t#o of ten patients inone trial had de%eloped leu+emia resulting from the insertion of the gene5carrying retro%irus near an

oncogene. -n 0BB


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Manufacture large $uantities of DNA, and someho# pac+age it to induce the target cells to accept it,

either as an addition to one of the original 03 chromosomes, or as an independent 0th human artificial

chromosome.

Germline

Germline engineering in%ol%es changing genes in eggs, sperm, or %ery early embryos. his type of

engineering is inheritable, meaning that the modified genes #ould appear not only in any children that

resulted from the procedure, but in all succeeding generations. Germline engineering is contro%ersialdue to the ability to change the %ery underlying nature of humanity in fundamental #ays according

simply to personal %alues of the indi%iduals undergoing or performing the change on their children.

6ugenics and genetic engineering of humans has been recei%ed badly throughout the scientificcommunity due to its history in the early and mid 0Bth century.

Uses of Human Genetic Engineering

here are t#o types of human genetic engineering, negati%e and positi%e. he former remo%es geneticdisorders and the latter enhances.

egati!e genetic engineering "cures and treatments#

hen treating problems that arise from genetic disorder, one solution is gene therapy, also +no#n asnegati%e genetic engineering. A genetic disorder is a condition caused by the genetic code of the

indi%idual, such as spina bifida or autism." hen this happens, genes may be epressed in

unfa%orable #ays or not at all, and this generally leads to further complications.

he idea of gene therapy is that a non5pathogenic %irus or other deli%ery systems can be used to insert

into DNAHa good copy of the geneHinto cells of the li%ing indi%idual. he modified cells #oulddi%ide as normal and each di%ision #ould produce cells that epress the desired trait. he result #ouldbe that he/she #ould then ha%e the ability to epress the trait that #as pre%iously absent, at least

partially. his form of genetic engineering could help alle%iate many problems, such as diabetes, cystic

fibrosis, or other genetic diseases.

$ositi!e genetic engineering "enhancement#

he potential of genetic engineering to cure medical conditions opens the $uestion of eactly #hat such

a condition is. &ome %ie# aging and death as medical conditions and therefore potential targets for

engineering solutions. hey see human genetic engineering potentially as a +ey tool in this (see life

etension). he difference bet#een cure and enhancement from this perspecti%e is merely one ofdegree. heoretically genetic engineering could be used to drastically change people's genomes, #hich

could enable people to regro# limbs and other organs, perhaps e%en etremely comple ones such as

the spine.

-t could also be used to ma+e people stronger, faster, smarter, or to increase the capacity of the lungs,

among other things. -f a gene eists in nature, it could be brought o%er to a human cell. -n this %ie#,there is no $ualitati%e difference (only a $uantitati%e one) bet#een, for instance, a genetic inter%ention

to cure muscular atrophy, and a genetic inter%ention to impro%e muscle function e%en #hen those

muscles are functioning at or around the human a%erage (since there is also an a%erage muscle function


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for those #ith a particular type of dystrophy, #hich the treatment #ould impro%e upon).

7thers feel that there is an important distinction bet#een using genetic technologies to treat those #ho

are suffering and to ma+e those #ho are already healthy superior to the a%erage. hough theory andspeculation suggest that genetic engineering could be used to ma+e people stronger, faster, smarter, or

to increase lung capacity, the AAA& report finds that there is little e%idence that this can currently be

done #ithout %ery unsafe and therefore unethical human eperiments. ?ecause different cells ha%edifferent tas+s, changing one cell to do a different :ob #ill not only affect that one tas+, it can affect

many others too.

%ontro!ersy &!er Human Genetic Engineering

Ethics

he genetic engineering of humans has raised many contro%ersial ethical issues. hile negati%e genetic

engineering (gene therapy) does indeed raise a debate, the use of genetic engineering for human

enhancement arouses the strongest feelings on both sides."

Genetic engineering is tested on animals, often including primates. &ome animal rights acti%ists find

this inhumane."

Genetic modification of embryos can pose an ethical $uestion about the rights of the baby. 7ne belief is

that e%ery fetus should be free to not be genetically modified. 7thers belie%e that parents hold the rightsto change their unborn children.


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'orms of Genetic Engineering

Genetic engineering can enable the transport of genes bet#een unrelated (transgenesis) or related(cisgenesis) organisms that #ould other#ise be unable to occur naturally, due to differences in anatomy

or the incorrespondence bet#een the DNA structures." his form of genetic engineering can produce

unpredictable results to the genome of the organisms, and can be related to those mutation processes."

Ad!antages

he modification of the DNA structures of agricultural crops can increase the gro#th rates and e%en

resistance to different diseases caused by pathogens and parasites.!B" his is etremely beneficial as it

can greatly increase the production of food sources #ith the usage of fe#er resources that #ould bere$uired to host the #orld's gro#ing populations. hese modified crops #ould also reduce the usage of

chemicals, such as fertili;ers and pesticides, and therefore decrease the se%erity and fre$uency of the

damages produced by these chemical pollution." Domesticated animals can undergo the same

mechanism. Genetic engineering can also increase the genetic di%ersity of species populations,especially those that are classified as being endangered. -ncrease in genetic di%ersity #ould enabled

these organisms to e%ol%e more efficiently that #ould allo# better adaptation to the ecosystems they

inhabit. -t #ould also reduce the %ulnerability of certain diseases produced by pathogens, as #ell asdecrease the ris+ of inbreeding that #ould produce infertile youths. Genetic engineering can be

performed to increase to the efficiency of the ecosystem ser%ices pro%ided by the other organisms.!!"

4or eample, the modification of a tree's genes could perhaps increase the root systems of theseorganisms reduce the damage produced by flood phenomena through flood mitigation.

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