Biotechnology (Genetic Engineering) - customizing genes in one or more species to suit human desires.

A. Recombinant DNA - the process of combining DNA from one individual with another. How its done: See digital handout on my website.

B. 1. isolate the desired gene and cut it out with restriction enzyme ; a genetic scissors that cuts DNA at a specific site, producing sticky ends.

2. cut open a section of bacterial DNA called a plasmid, with the same restriction enzyme mentioned above.

3. splice the desired gene into the plasmid, connecting the sticky ends together with an enzyme called ligase.

4. Reintroduce the modified plasmid into a bacterial cell

5. Let bacteria reproduce; only now every time the bacteria reproduces, it will make more copies of the desired gene within it. More importantly, the bacteria is manufacturing the desired protein.

6. Harvest the protein produced by the bacteria

NOTE: SOMETIMES THE PROTEIN IS HARVESTED FROM THE BACTERIA. OTHER TIMES, THE ALTERED GENE IS PUT DIRECTLY INTO THE ORGANISM. REFER TOTHE NOTES ON GENE THERAPY AT THE END OF THIS STUDY GUIDE AND READINGS DONE IN CLASS B. Commercial applications of recombinant DNA.

1. Insulin - in production since 1982. Treatment for diabetes

2. Interferon - a natural defense against viral infections and certain types of cancer. Formerly available in small amounts; can now be produced in large amounts.

3. Factor 8 and 9 - clotting factors that are needed by hemophiliacs

4. Human growth hormone - treatment for dwarfism

5. Flavor-Saver Tomato -designed to stay fresh longer

C. Other applications of genetic engineering

l. Transgenic animals (chimeras) - splicing the DNA of one animal species with another. See cartoon below.

a. pigs and cows having human genes in them; act as living human protein factories. May even produce human organs in the near future.

2. Improved agricultural yields, producing more food using less resources, and

resistance to disease.

3. Vaccines for diseases such as AIDS

4. Gene therapy - fixing the defective gene that causes a specific disease and then inoculating the individual with doses of the healthy gene.

a. Cystic Fibrosis patients inhale a genetically engineered virus to replace defective cells in their b. A cell killer gene known as p53 is introduced into cancer cells to stop

them from dividing * John Sulston's work on the C. elegans worm led to the discovery of p53 in people!

After flicking on the light Professor Zurkowitz is caught off guard by the overnight success of his efforts to cross-breed flying fish and piranha

Young Victor Frankenstein stays after school

Biotechnology Products Today, bacteria, plants, and animals are genetically engineered- that is, genetically altered to make biotechnol- ogy products (Fig. 16.3). Organisms that have had a foreign gene inserted into them are called transgenic organisms [L. trans, across, through, and Gk., -genie, producing].

Transgenic Bacteria Recombinant DNA technology is used to produce transgenic bacteria, which are grown in huge vats called bioreactors. The gene product is collected from the medium. Biotechnol- ogy products now on the market that are produced by bacte- ria include insulin, human growth hormone, t-PA (tissue plasminogen activator), and hepatitis B vaccine. Transgenic bacteria have many other uses as well. Some have been pro- duced to promote the health of plants. For example, bacteria that normally live on plants and encourage the formation of ice crystals have been changed from frost-plus to frost-minus bacteria. Also, a bacterium that normally colonizes the roots of corn plants has now been endowed with genes (from another bacterium) that code for an insect toxin. The toxin protects the roots from insects.

Bacteria can be selected for their ability to degrade a particular substance, and this ability can then be enhanced by genetic engineering. For instance, naturally occurring bacteria that eat oil can be genetically engineered to do an even better job of cleaning up beaches af ter oil spills.

Figure 16.3 Biotechnology products. Products such as clotting factor VIII, which is administered to hemophiliacs, can be made by transgeni c bacteria, plants, or animals. After being processed and packaged , the product is sold commercially.

Industry has found that bacteria can be used as biofilters to prevent airborne chemical pollutants from being vented into the air. Bacteria can also remove sulfur from coal be- fore it is burned and help clean up toxic waste dumps. One such strain was given genes that allowed it to clean up lev- els of toxins that would have killed other strains. Further, these bacteria were given "suicide" genes that caused them to self-destruct when the job had been accomplished.

Organic chemica ls are often synthesized by having cat- alysts act on precursor molecules or by using bacteria to carry out the synthesis. Today, it is possible to go one step further and manipulate the genes that code for these en- zymes. For instance, biochemist s discovered a strain of bac- teria that is especially good at producing phenylalanine, an organic chemical needed to make aspartame, the dipeptide sweetener better known as NutraSweet. They isolated, al- tered, and formed a vector for the appropriate genes so that various bacteria could be genetically engineered to produce phenylalanine.

Many major mining companies already use bacteria to obtain various metals. Genetic engineering can enhance the ability of bacteria to extract copper, uranium, and gold from low-grade sources. Some mining companies are testing ge- netically engineered organisms that have improved bio- leaching capabilities.

Transgenic Plants Techniques have been developed to introduce foreign genes into immature plant embryos or into plant cells called proto- plasts that have had the cell wall removed. It is possible to treat protoplasts with an electric current while they are sus- pended in a liquid containing foreign DNA. The electric cur- rent makes tiny, self-sealing holes in the plasma membrane through which genetic material can enter. Protoplasts go on to develop into mature plants.

Foreign genes transferred to cotton, corn, and potato strains have made these plants resistan t to pests because their cells now produce an insect toxin. Similarly, soybeans have been made resistant to a common herbicide. Some corn and cotton plants are both pest- and herbicide-resis tant. These and other genetically engineered crops that are ex- pected to have increased yield are now sold commercially.

Plants are also being engineered to produce human proteins, such as hormones, clotting factors, and antibodies, in their seeds. One type of antibody made by corn can de- liver radioisotopes to tumor cells, and another made by soy- beans can be used to treat genital herpes.

Transgenic Animals Techniques have been developed to insert genes into the eggs of animals. It is possible to microinject foreign genes into eggs by hand, but another method uses vortex mixing. The eggs are placed in an agitator with DNA and silicon-carbide

needles, and the needles make tiny holes through which the DNA can enter. When these eggs are fertilized, the resulting offspring are transgenic animals. Using this technique, many types of animal eggs have taken up the gene for bovine growth hormone (bGH). The procedure has been used to produce larger fishes, cows, pigs, rabbi ts, and sheep.

Gene pharming, the use of transgenic farm anima ls to produce pharmaceuticals, is being pursued by a number of firms. Genes that code for therapeutic and diagnostic

human gene \

....,.

egg

proteins are incorporated into an animal's DNA, and the pro- teins appear in the animal's milk. Plans are underway to pro- duce drugs for the treatment of cystic fibrosis, cancer, blood diseases,and other disorders by this method. Figure 16.4 out- lines the procedure for producing transgenic mammals: DNA containing the gene of interest is injected into donor eggs. Following in vitro fertiliza tion, the zygotes are placed in host females, where they develop. After female offspring mature, the product is secreted in their milk.

Cloning Transgenic Animals For many years, it was believed that adult vertebrate ani- mals could not be cloned. Although each cell contains a copy of all the genes, certain genes are turned off in mature, specialized cells. Cloning of an adult vertebrate requires that all the genes of an adult cell be turned on again if de- velopment is to proceed normally. This had long been thought impossible.

In 1997, however, Scottish scientists announced that they had produced a cloned sheep called Dolly. Since then, calves and goats have also been cloned, as described in Figure 16.4. After enucleated eggs from a donor are microinjected with 2n nuclei from the same transgenic animal, they are coaxed to begin development in vitro. Development contin- ues in host females until the clones are born. The offspring are clones because all have the genotype and phenotype of the adult that donated the 2n nuclei. In a procedure that pro- duced cloned mice, the 2n nuclei were taken from corona ra- diata cells. Corona radiata cells are those that cling to an egg after ovulation occurs. Now that scientists have a way to clone animals, this procedure will undoubtedly be used rou- tinely to procure biotechnology products.

adult cells 2n nuclei

microinject ion of human gene

:= de_v_elop "' hoot go"

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milk transgen ic goat

egg donor

enucleated eggs

Animal Organs as Biotechnology Products The Health Focus on the next page discusses how it may be .. .. .. ..

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possible for genetically engineered pigs to serve as a source of organs for human transplant operations. Alternatively, scientists are learning how to stimulate human cells to con- struct organs in the laboratory.

Genetically engineered bacteria,plants,and animals are used to make biotechnology products. Procedures have also been developed to clone these animals. Organs made in the laboratory are also biotechnology products.

cloned transgenic goats

milk Figure 16.4 Transgenic mammals. A genetically engineered egg develops in a host to create a transgenic goat that produces a biotechnology product in its milk. Nuclei from the transgenic goat are transferred into donor eggs, which develop into cloned transgenic goats.

i

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development within host goats

Health Focus

Organs for Transplant

lthough it is now possible to trans- plant various organs, there are not

enough human donors to go around. Thousands of patients die each year while waiting for an organ. It's no wonder,then, that scientists are suggesting we get or- gans from a source other than other hu- mans. Xenotransplantation [Gk. xenos, strange, foreign) is the use of animal or- gans instead of human organs in trans- plant patients. You might think that apes, such as the chimpanzee or the baboon, would be a scientifically suitable species for this purpose. But apes are slow breed- ers and probably cannot be counted on to supply all the organs needed. Also, many people might object to using apes for this purpose. In contrast, animal husbandry has long included the raising of pigs as a meat source, and pigs are prolific. A fe- male pig can become pregnant at six months of age and can have two litters a year,each averaging about ten offspring.

Ordinarily, the human body would vi- olently reject transplanted pig organs. Ge- netic engineering, however, can make these organs less antigenic. Scientists have produced a strain of pigs whose organs would most likely, even today, survive for a few months in humans. They could be used to keep a patient alive until a human organ was available. The ultimate goal is to make pig organs as widely accepted by humans as type 0 blood. (A person with type 0 blood is called a universal donor because the red blood cells carry no A or B antigens.)

As xenotransp lantation draws near, other concerns have been raised. Some ex- perts fear that animals might be infected with viruses, akin to Ebola virus or the "mad cow" disease virus. After infecting a transplant patient, these viruses might spread into the general populace and be- gin an epidemic. As an indication of this possibility, scientists believe that HIV was spread to humans from monkeys when humans ate monkey meat. Advocates of using pigs for xenotransplantation point out that pigs have been around humans for centuries without infecting them with any serious diseases.

An alternative to xenotransplantation

come from humans or other animals. Now, however, tissue engineering is demonstrating that it is possible to make some bioartificial organs-hybrids cre- ated from a combination of living cells and biodegradable polymers. Presently, lab-grown hybrid tissues are on the mar- ket. For example, a product composed of skin cells growing on a polymer is used to temporarily cover the wounds of burn pa- tients. Similarly,damaged cartilage can be replaced with a hybrid tissue produced af- ter chondrocytes are harvested from a pa- tient. Another connective tissue product made from fibroblasts and collagen is available to help heal deep wounds with- out scarring. Soon to come are a host of other products, including replacement corneas, heart valves, bladder valves, and breast tissue.

Tissue engineers have also created cel- lular implants-cells producing a useful product encapsulated within a narrow plastic tube or a capsule the size of a dime or quarter. The pores of the container are large enough to allow the product to dif- fuse out but too small for immune cells to

enter and destroy the cells. An implant whose cells secrete natural pain killers will survive for months in the spinal cord and can be easily withdrawn when desired. A "bridge to a liver transplant" is a bedside vascular apparatus. The patient's blood passes through porous tubes surrounded by pig liver cells. These cells convert toxins in the blood to nonpoisonous substances.

The goal of tissue engineering is to produce fully function ing organs for transplant. After nine years, a Harvard Medical School team headed by Anthony Ata la has produced a working urinary bladder in the laboratory (Fig. 16A). After testing the bladder in laboratory animals, the Harvard group is ready to test it in hu- mans whose own bladders have been damaged by accident or disease, or will not function properly due to a congenital birth defect. Another group of scientists has been able to grow arterial blood ves- sels in the laboratory. Tissue engineers hope that they will one day produce larger internal organs such as the liver or kidney.

also exists. Just a few years ago, scientists believed that transplant organs had to

Figure 16A Tissue engineering. This urinary bladder was made in the laboratory by tissue engineering.

The Human Genome Project

A genome is all the genetic information of an individ ual or a species. The Human Genome Project has two goals: (1) to con- struct a map that shows the sequence of base pairs along our chromosomes, and (2) to construct a map that shows the sequence of genes a long the human chromosomes.

The Base Sequence Map Researchers have now completed the first goal. They know the sequence of the three billion base pairs, one af ter the other, along the length of our chromosomes. It took some fif teen years for researchers to com- plete this monumental task. Two rival groups have been at work on the project. The International Human Genome Se- quencing Consortium, which consists of laboratories in many different countries, depends on the support of public funds, in- cluding substantial contributions from the United States government. On the other hand, Celera Genomics, a private company that is supported by a pharmaceutica l firm, has been sequencing the genome for only a few years. These competing groups used slightly different techniques, but their data match.

Even though we now know the se- quence of bases in the human genome, much work still needs to be done to make sense out of what we have discovered. We have found that there is little difference be- tween the sequence of our bases and other organisms whose DNA sequences are also known. From this we can conclude that we share a large number of genes with much simpler organisms, including bacteria! It's possible that eventua lly we will discover that our uniqueness is due to the regulation of these genes.

The Genetic Map The genetic ma p tells the location of genes along each chromosome. Figure 16.5shows the loci of significan t mu ta nt genes on hu- man chromosome 17. Many genes have had their loci determined. Still, we do not

know the sequence of all the genes on any particular chromosome.

Completing the chromosomal genetic map should accelerate now that the base- pair

sequence map is complete. Research- ers need only know a short sequence of bases in a gene of interest in order for the computer to search the genome for a match. Then, the computer will tell the re- searcher where this gene is located.

A question still being hotly debated is the total number of human genes. Much of our DNA consists of nucleotide repeats that do not code for a protein. So far, researchers have found only 30,000 genes that code for proteins. This number seems terribly low; that is, a round worm has 20,000 genes, so a human, which is certainly more complex than a roundworm, should have many more genes. Some researchers think more genes are yet to be identified. Others, be- lieving they have found most of our genes, speculate that each of these genes could code for about three proteins,simply by us- ing different combinations of exons.

As discussed in the Health Focus on page 274, researchers are hopeful that map- ping the human chromosomes will help them not only discover mutant genes for many more human disorders but also de- velop medicines to treat these disorders. In addition, it may be possible to locate genes suitable for gene therapy to cure human ill- nesses or enhance a phenotype. Such genes might be inserted into the egg before it is fertilized.

There are many ethical questions re- garding how our knowled ge of the human genome should be used. Therefore, it is imperative that everyone be educated about the human genome because in the end it is the public that will have to decide these issues.

One goal of the Human Genome Project has been met: to sequence the DNA bases of each chromosome. Geneticists are continuing to work on the second goal:to map the loci of genes on each chromosome. This information should contribute to the health of human beings.

retinitis pigmentosa

cataract

diabetes susceptibility

cancer

deafness

Charcot-Marie- Tooth neuropathy

osteogenesis imperfecta

osteoporosis

anxiety-related personality traits

Alzheimer disease susceptibility

neurofibromatosis

leukemia

dementia

muscular dystrophy

breast cancer ovarian cancer

pituitary tumor

yeast infection susceptibility

growth hormone deficiency

myocardial infarction susceptibility

small-cell lung cancer

Figure 16.5 Genetic map of chromosome 17. This map shows the sequence of mutant genes that cause the diseases or conditions noted.

Health Focus

New Cures on the Horizon

Back in the 1980s, Leroy Hood couldn't get funding for the DNA sequencer

he was developing. Biologists didn't like the idea of just "collecting facts," and it took several years before an entrepreneur decided to fund the project. Without ever-better DNA sequencers, the Human Genome Project would never have com- pleted its monumental task of determin- ing the sequence of bases in our DNA.

Now that we know the sequence of all the bases in the DNA of all our chromo- somes, biologists all over the world be- lieve that this knowledge will result in rapid medical advances for ourselves and our children. At least four categories of improvement are expected: (1) Many more medicines will be available to keep us healthy; (2) medicines will be safer due to genome scans; (3) a longer life span, even to over 100 years, may become com- monplace; and (4) we will be able to shape the genotypes of our offspring.

First prediction: Many new medicines will be available. Genome sequence data will allow scien- tists to determine all the proteins that are

active only during development plus all those that are still active in adults.

Most drugs are either proteins or small chemicals that are able to interact with proteins. Many of these small chemi- cals target proteins that act as signals be- tween cells or within the cytoplasm of cells. Today's drugs were usually discov- ered in a hit-or-miss fashion, but now we can take a more systematic approach to finding effective drugs. For example, it is known that all receptor and signaling pro- teins start with the same ten-amino-acid sequence. Now, it is possible to scan the human genome for all genes that code for this sequence of ten amino acids, and thereby find all the signaling proteins. Thereafter,they must be tested.

In a recent search for a protein that makes wounds heal, researchers cultured skin cells with fourteen proteins (found by chance) that can cause skin cells to grow. Only one of these proteins made skin cells grow and did nothing else. They expect this protein to become an effective drug for conditions such as venous ulcers, which are skin lesions that affect many thousands of people in the United States. Such tests, leading to effective results, can be carried out with all the signaling pro- teins scientists will discover by scanning the human genome.

People's genotypes differ. We all have mutations that account for our various ill- nesses. Knowing each patient's mutations will allow physicians to match the right drugs to the particular patient.

Second prediction: Medicines will be safer due to genome scans. Genome scans will allow us to discover genetically different subgroups of the population. Physicians will be looking for two types of mutations in particular. One type is called single nucleotide polymor- phisms (SNPs, pronounced "snips"), in which individua ls differ by only one nu- cleotide. The other type of mutation is nucleotide repeats, in which the same

three bases, repeated over and over again, interrupt a gene and affect its ex- pression. It is not yet clear how many SNPs will be medically significant, but the pres- ent estimate is on the order of 300,000.

How will a physician be able to deter- mine which of the 300,000 SNPs and other types of mutations are in your genome? The use of a gene chip will quickly and ef- ficiently provide knowledge of your geno- type. A gene chip is an array of thousands of genes on one or several glass slides packaged together. After the gene chip is exposed to an individual's DNA, a techni- cian can note any mutant sequences pres- ent in the individual's genes. Soon a chip will be able to hold all the genes carried within the human genome.

Some disorders, such as sickle-cell dis- ease, are caused by a single SNP,but many disorders seem to require more than one, and possibly in different combinations. In one study, researchers found that a series of SNPs, numbered 1-12, were associated with the development of asthma. A partic- ular drug, called albuterol, was effective for patients with certain combinations and not others. This example and others show

that many diseases are polygenic, and that only a genome scan is able to detect which mutations are causing an individual to have the disease, and how it should be properly treated.

Genome scans are also expected to make drugs safer to take. A.s you know, many drugs potentially have unwanted side effects. Why do some people and not others have one or more of the side ef- fects? Most likely, because people have different genotypes. It is expected that a physician will be able to match patients to drugs that are safe for them on the basis of their genotypes.

Biologists also hope that gene chips will single out the specific oncogenes and mutated tumor-suppressor genes that cause the various types of cancer.The pro- tein products of these genes will become targets against which chemists can try to develop drugs. If so, the current methods of treating cancer-surgery, radiation, and chemotherapy to kill all dividing cells-will no longer be necessary.

Third prediction: A longer and healthier life will be yours. Genome sequence data may allow scien- tists to determine which genes enable people to live longer. Investigators have already found evidence for genes that ex- tend the life span of animals such as roundworms and fruit flies. The sequenc- ing of the human genome makes it possi- ble for scientists to find such genes in hu- mans also.

For example, we know that the pres- ence of free radicals causes cellular mole- cules to become unstable and cells to die. Certain genes are believed to code for an- tioxidant enzymes that detoxify free radi- cals. It could be that human beings with particular forms of these genes have more efficient antioxidant enzymes, and there- fore live longer. If so, researchers will no doubt be able to locate these genes and also others that promote a longer life.

Consider, too, that natural selection favors phenotypes that result in the great- est number of fertile offspring in the next generation. Since children are usually born to younger individuals, natural selec- tion is indifferent to genes that protect the body from the deleterious effects of aging. Researchers can possibly find such genes, however,in individuals who have a long life span. Use of these genes would possibly oppose a destiny, determined so far only by evolution.

Possible stem cell therapy has gener- ated much interest of late. Stem cells are embryonic cells and also some adult cells, such as those in red bone marrow,that are nondifferentiated. These cells have the potential to become any type of tissue, depending on which signaling molecules are used. Genome sequence data will eventually give scient ists knowledge of all the signaling molecules humans possess. Stem cells could also be subjected to gene therapy in order to correct any defective genes before scientists use them to create the tissues or organs of the body. Use of these tissues and organs to repair and/or

replace worn-out structures could no doubt expand the human life span.

Fourth prediction: You will be able to design your children. Genome sequence data will be used to identify many more mutant genes that cause genetic disorders than are presently known. In the future,it may be possible to cure genetic disorders before the child is born by adding a normal gene to any egg that carries a mutant gene. Or an artificial chromosome,constructed to carry a large number of corrective genes, could auto- matically be placed in eggs. In vitro fertil- ization would have to be utilized in order to take advantage of such measures for curing genetic disorders before birth.

Genome sequence data can also be used to identify polygenic genes for traits such as height, intelligence, or behavioral characteristics. A. couple could decide on their own which genes they wish to use to enhance a child's phenotype. In other words, the sequencing of the human genome may bring about a genet ically just society, in which all types of genes would be accessible to all parents.

Figure 16.6 Ex vivo gene therapy in humans. Bone marrow stem cells are withdrawn from the body, an RNA retrovirus is used to insert a normal gene into them, and they are returned to the body.

16.4 Gene Therapy Gene therapy is the insertion of genetic material into human cells for the treatment of a disorder. It includes procedures that give a patient healthy genes to make up for faulty genes, as well as the use of genes to treat various other hu- man illnesses, such as cardiovascular disease and cancer. Gene therapy includes both ex vivo (outside the body) and in vivo (inside the body) methods.

Ex Vivo Gene Therapy Figure 16.6 describes the methodology for treating children who have SCID (severe combined immunodeficiency). These children lack the enzyme ADA (adenosine deami- nase), which is involved in the maturation of T and B cells. In order to carry out gene therapy, bone marrow stem cells are removed from the blood and infected with an RNA retrovirus that carries a normal gene for the enzyme. Then the cells are returned to the patient. Bone marrow stem cells are preferred for this procedure because they divide to pro- duce more cells with the same genes. Patients who have undergone this procedure show significantly improved im- mune function associa ted with a sustained rise in the level of ADA enzyme activity in the blood.

Among the many gene therapy trials, one is for the treatment of familial hypercholesterolemia , a condition that develops when liver cells lack a receptor protein for remov- ing cholesterol from the blood. The high levels of blood cho- lesterol make the patient subject to fatal heart attacks at a young age. A small portion of the liver is surgically excised and then infected with a retrovirus containing a normal gene for the receptor before being returned to the patient.

Several patients have experienced lowered serum choles- terol levels following this procedure.

In Vivo Gene Therapy Cystic fibrosis patients lack a gene that codes for the trans- membrane carrier of the chloride ion. They often die due to numerous infections of the respiratory tract. In gene therapy trials, the gene needed to cure cystic fibrosis is sprayed into the nose or delivered to the lower respiratory tract by adeno- viruses or by the use of liposomes, microscopic vesicles that spontaneously form when lipoproteins are put into a solu- tion. Investigators are trying to improve uptake, and are also hypothesizing tha t a combination of all three vectors might be more successful.

Genes are being used to treat medical conditions such as poor coronary circulation. It has been known for some time that VEGF (vascular endothelial growth factor) can cause the growth of new blood vessels. The gene that codes for this growth factor can be injected alone or within a virus into the heart to stimula te branching of coronary blood ves- sels. Patients report that they have less chest pain and can run longer on a treadmill.

Gene therapy is increasingly used as a part of cancer therapy. Genes are being used to make healthy cells more tolerant of chemotherapy, and to make tumors more vulner- able to chemotherapy. The gene p53 brings about apoptosis, and there is much interest in introducing it into cancer cells, and in that way killing them off.

Both ex vivo and in vivo methods of gene therapy are playing a role in curing illnesses.

3. Recombinant DNA carries normal gene into genome.

4. Return genetically engineered cells to patient.

1. Remove bone marrow

stem cells.

2. Use retroviruses to infect bone marrow stem cells with normal gene.

reverse transcription

recombinant DNA

recombinant RNA

gene

defective gene