gene transfer 1

7/30/2019 Gene Transfer 1

1/9

TRANSGENESISTransgenesisThe use of recombinant DNA techniques to introduce new characters (ie.genes) into organisms (including humans) that were not present previously.The term transgenic animal refers to an animal in which there has been adeliberate modification of the genome, in contrast to spontaneous mutation.Foreign DNA is introduced into the animal, using recombinant DNAtechnology, and then must be transmitted through the germ line so thatevery cell, including germ cells, of the animal contain the same modifiedgenetic material.If the germ cell line is altered, characters will be passed on to succeedinggenerations in normal reproduction.If the somatic cell line alone is altered, only the organism itself will beaffected, not its offspring.Transgenesis may involve whole organisms, rather than individual cells,and there may be in vivoalteration of body function.One use of transgenesis is

gene therapywhich is the alteration of the

genetic make-up of of an individual organism in an attempt to correct aninborn error of metabolism, ie. cure inherited diseases. But this is generallyonly carried out with somatic cells and, therefore, will only affect onegeneration.Do not confuse transgenesis with cloning which is the production ofidentical copies of molecules, cells or whole organisms. Cloning does notnecessarily involve gene manipulation.

Historical backgroundDuring the 1970s, the first chimeric mice were produced (Brinster, 1974).The cells of two different embryos of different strains were combinedtogether at an early stage of development (eight cells) to form a singleembryo that subsequently developed into a chimeric adult, exhibitingcharacteristics of each strain. Subsequently this was done with otheranimals, eg. sheep and goat ----> "geep" (1982).


2/9

Transgenesis was first described in 1981 (Gordon and Ruddle, 1981)using DNA microinjection of mice ova. Various other species such as rats,rabbits, sheep, pigs, birds, and fish soon followed.It has been gaining application among biotechnologists since thedevelopment of transgenic "super mice" in 1982 and the development ofthe first mice to produce a human drug, tPA (tissue plasminogen activatorto treat blood clots), in 1987.Two other main techniques have been developed: those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossleret al., 1986).

Uses of transgenic organisms: in toxicology: as responsive test animals (detection of toxicants); in mammalian developmental genetics; to introduce human genes into other organisms (particularly human)

for the study of disease processes; in molecular biology, the analysis of the regulation of gene

expression;

in the pharmaceutical industry, the production of humanpharmaceuticals in farm animals ("pharming"); targeted productionof pharmaceutical proteins, drug production and product efficacytesting;

in biotechnology: as producers of specific proteins; genetically engineered hormones to increase milk yield, meat

production; genetic engineering of livestock in agriculture affectingmodification of animal physiology and/or anatomy; cloningprocedures to reproduce specific blood lines;

to speed up the introduction of existing characters into a strain/breed

for improvement and modification; developing animals specially created for use in xenografting, ie.

modify the antigenic make-up of animals so that their tissues andorgans can be used in transfusions and transplants.

Procedure for transgenesisThe inserted DNA is known as the transgene.


3/9

Conventional recombinant DNA techniques are used to construct thetransgene so that the desired gene product will be expressed in the desiredlocation. Typical transgenes contain nucleotide sequences that correspondto the gene of interest, with all the components necessary for efficient

expression of the gene, including a transcription-initiation site, the 5'untranslated region, a translation-initiation codon, the coding region, a stopcodon, the 3' untranslated region, a polyadenylation site and a promoter.Different promoters can be used to cause gene expression in all tissues ofthe body (non-specific) or only in specific tissues:eg.

PROMOTER GENE EXPRESSION IN(beta)-actin promoter many tissues of the transgenic animalsimian virus 40 T antigen promoter many tissues of the transgenic animaladipocyte P2 promoter fat cellsmyosin light-chain promoter muscleamylase promoter acinar pancreasinsulin promoter islets of Langerhans beta cellsbeta-lactoglobin promoter mammary glands

In pharming expression in the mammary glands is usually desired as thisleads to the appearance of the product in the milk of the animal - veryconvenient.

Introduction of exogenous DNA into animal cellsThe three principal methods used for the creation of transgenic animals areDNA microinjection, embryonic stem cell-mediated gene transfer and

retrovirus-mediated gene transfer.1. DNA microinjectionThis method involves the direct microinjection of a chosen gene construct(a single gene or a combination of genes) from another member of thesame species or from a different species, into the pronucleus of a fertilizedovum. It is one of the first methods that proved to be effective in mammals(Gordon and Ruddle, 1981) which are the most difficult of all cells togenetically manipulate. The introduced DNA may lead to the over- or

under-expression of certain genes or to the expression of genes entirelynew to the animal species. The DNA construct (usually about 100 to 200


4/9

copies in 2 pl of buffer) is introduced by microinjection through a fine glassneedle into the male pronucleus - the nucleus provided by the spermbefore fusion with the nucleus of the egg. The diameter of the egg is 70 mand that of the glass needle is 0.75 m; the experimenter performs the

manipulations with a binocular microscope at a magnification of 200 x.Theinsertion of DNA is, however, a random process, and there is a highprobability that the introduced gene will not insert itself into a site on thehost DNA that will permit its expression. The manipulated fertilized ovum istransferred into the oviduct of a recipient female, or foster mother that hasbeen induced to act as a recipient by mating with a vasectomized male.Microinjection is the commonest method at present and is generally moresuccessful with laboratory animals than farm animals.The efficiency of microinjection is quite low:

Animalspecies

Number ofova

injectedNumber ofoffspring

Number oftransgenicoffspring

rabbit 1907 218 (11.4%) 28 (1.5%)sheep 1032 73 (7.1%) 1 (0.1%)

pig 2035 192 (9.4%) 20 (1.0%)Figures in parentheses are percent efficiency compared to original number

of ova injected.(after Hammeret al., 1985)

2. Embryonic stem cell-mediated gene transferThis method involves prior insertion of the desired DNA sequence byhomologous recombination into an in vitro culture of embryonic stem (ES)cells. Stem cells are undifferentiated cells that have the potential todifferentiate into any type of cell (somatic and germ cells) and therefore togive rise to a complete organism. These cells are then incorporated into anembryo at the blastocyst stage of development. The result is a chimericanimal. ES cell-mediated gene transfer is the method of choice for geneinactivation, the so-called knock-out method.This technique is of particular importance for the study of the genetic

control of developmental processes. This technique works particularly well


5/9

in mice. It has the advantage of allowing precise targeting of definedmutations in the gene via homologous recombination.

3. Retrovirus-mediated gene transferTo increase the probability of expression, gene transfer is mediated bymeans of a carrier or vector, generally a virus or a plasmid. Retrovirusesare commonly used as vectors to transfer genetic material into the cell,taking advantage of their ability to infect host cells in this way. Offspringderived from this method are chimeric, i.e., not all cells carry the retrovirus.Transmission of the transgene is possible only if the retrovirus integratesinto some of the germ cells.For any of these techniques the success rate in terms of live birth ofanimals containing the transgene is extremely low. Providing that thegenetic manipulation does not lead to abortion, the result is a firstgeneration (F1) of animals that need to be tested for the expression of thetransgene. Depending on the technique used, the F1 generation may resultin chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations untilhomozygous transgenic animals are obtained and the transgene is presentin every cell. At this stage embryos carrying the transgene can be frozen

and stored for subsequent implantation.There is also fusion of host cells with membranous vesicles (eg. liposomes)containing DNA.

(Plant cells can be modified using, eg. tobacco mosaic virus or the Tiplasmid ofAgrobacterium tumefaciens.)

Some examples of the use of transgenic organismsStudying diseaseTransgenic animals have been used for simulating diseases and testingnew therapies, eg. cardiovascular and neurodegenerative diseases. Animal


6/9

models provide an opportunity to test methods for the prevention or delayof disease in humans. Some examples:Genetic alteration Method of alteration Human disease

equivalentIntroduction of mutant collagengene into wildtype mice Nuclear microinjection ofinducible minigene OsteogenesisimperfectaInactivation of mouse geneencoding hypoxanthine-guaninephosphoribosyl transferase(HPRT)

Insertion of retrovirus intoHPRT locus in embryonicstem cells

HPRT deficiency

Mutation at locus for X-linkedmuscular dystrophy Male mutagenesis followedby identification of female

carriersX-linked musculardystrophy

Introduction of activatedhumanrasand c-myconcogenes Nuclear microinjection ofinducible minigene Induction ofmalignancyIntroduction of mutant (Z)allele ofhuman alpha -1-antitrypsin gene Microinjection of DNAfragment bearing mutant

allele

Neonatal hepatitis

Introduction of HIV tatgene Microinjection of DNAfragment Kaposi's sarcoma

Introduction of beta-globin sicklegene Microinjection Sickle-cellanaemiaIntroduction of mouse renin gene Microinjection HypertensionIntroduction of (beta)-amyloidprotein precursor (APP gene) Microinjection Alzheimer'sdisease

Improving plantsTransgenic methods have now been developed for a number of importantcrop plants such as rice, cotton, soybean, oilseed rape and a variety ofvegetable crops like tomato, potato, cabbage and lettuce. New plantvarieties have been produced using bacterial or viral genes that confer

tolerance to insect or disease pests and allow plants to tolerate herbicides,


7/9

making the herbicide more selective in its action against weeds andallowing farmers to use less herbicide.

A new variety of cotton, for example, has been developed that uses a genefrom the bacterium Bacillus thuringiensisto produce a protein that isspecifically toxic to certain insect pests including bollworm, but not toanimals or humans. (This protein has been used as a pesticide spray formany years.) These transgenic plants should help reduce the use ofchemical pesticides in cotton production, as well as in the production ofmany other crops which could be engineered to contain the Bacillusthuringiensisgene. In another case, a gene from the potato leaf-roll virushas been introduced into a potato plant, giving the plant resistance to thisserious potato disease.Transgenic technologies are now being used to modify other importantcharacteristics of plants such as the nutritional value of pasture crops or theoil quality of oilseed plants like linseed or sunflower.

Improving livestockThe main aim in using transgenic technology in animal agriculture is toimprove livestock by altering their biochemistry, their hormonal balance or

their important protein products. Scientists hope to produce animals thatare larger and leaner, grow faster and are more efficient at using feed,more productive, or more resistant to disease. Examples of transgenicbreeding programs include:

producing faster-growing and leaner pigs that use food moreefficiently and resist common diseases

breeding transgenic sheep that grow better wool without needingdietary supplements of sulphur-containing amino acids.

The welfare of the animals, including any changes in metabolism that maycause health problems, is an important consideration in these programmes.Both researchers and regulatory bodies also examine closely any changesin the composition of meat or other products that will ultimately be eaten.

Advantages of transgenesis over selective breeding for animals andplants


8/9

Transgenic technology is an extension of agricultural practices that havebeen used for centuries: selective breeding and special feeding orfertilization programmes. It may reduce or even replace the large-scale useof pesticides and long-lasting herbicides. When fully developed, it would

offer a number of advantages over traditional methods.

Compared with traditional methods, transgenic breeding is: More specific scientists can choose with greater accuracy the trait

they want to establish. The number of additional unwanted traits canbe kept to a minimum.

Faster establishing the trait takes only one generation comparedwith the many generations often needed for traditional selectivebreeding, where much is left to chance.

More flexible

traits that would otherwise be unavailable in someanimals or plants may be achievable using transgenic methods.

Less costly much of the cost and labour involved in administeringfeed supplements and chemical treatments to animals and cropscould be avoided.

PharmingMany valuable pharmaceutical products can now be made using transgenicanimals such as mice, rabbits, sheep, goats, pigs and cows. Often theproduct conveniently appears in the milk of the animal. Transgenic plantscan also be used to make pharmaceuticals.Some examples:

factor VIII blood clotting factor factor IX blood clotting factor fibrinogen blood clotting factor lactoferrin as an infant formula additive haemoglobin as a blood substitute human protein C anticoagulant alpha-1-antitrypsin (AAT) for treatment of AAT deficiency tissue plasminogen activator (tPA) cystic fibrosis transmembrane conductance regulator (CFTR)

for treatment of CF insulin for diabetes treatment growth hormones for treatment of deficiencies

monoclonal antibodies

vaccines (antigens)


9/9

gene transfer 1

Documents