CHAPTER 19: Genetic & Reproductive Technologies

Presentors: Fernando, Camille Mamawan, Jinesh Manuel, Czarlene Pascasio, Jaia Vallejos, Marie Deguchi, Erika Estrada, Patrick Evangelista, Marix Galicia, Klarizza Ignacio, Pearl Legaspi, Apple Maralit, Pauline

BSMT 3EF Credits to: Human Genetics by H.R. Lewis

Introduction
Biotechnology is the use or alteration of cells or biological molecules for specific applications A transgenic organism has DNA from different species Recombinant DNA comes from more than one type of organism Both are possible because of the universality of the genetic code
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Amplifying DNA
Polymerase chain reaction (PCR)
Works on DNA molecules outside of cells Replicates sequence millions of times

Recombinant DNA technology

Amplifies DNA within cells often using sequences from other organisms

PCR
Consists of a repetition of three basic steps:
1. Denaturation: Heat is used to separate the two strands of target DNA 2. Annealing: Two short DNA primers bind to the DNA at a lower temperature 3. Extension: The enzyme Taq1 DNA polymerase adds bases to the primers

All this is done in a thermal cycler Copies of DNA accumulate exponentially

PCR
STEP Denaturation Annealing Extension TEMPERATURE 90-96degC 40-68degC 70-75degC TIME 20 secs 20 secs 30 secs

Applications of PCR:
> structural analysis of DNA > cloning > disease detection (HIV, SARS, H5N1, Meningococcus, Legionellosis, analysis of resistant genes MRSA and VRE > mutation analysis > mapping of DNA

Recombinant DNA Technology

Recombinant DNA technology is also known as gene cloning It began in 1975 when molecular biologists convened to discuss the safety and implications of this new technology However, it turned out to be safer than expected
It also spread to industry faster and in more diverse ways than imagined
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Creating Recombinant DNA Molecules

Manufacturing recombinant DNA requires restriction enzymes that cut donor and recipient DNA at the same sequence These enzymes cut DNA at sites that are palindromic The cutting action of many of these enzymes generate single-stranded extensions called sticky ends

Isolating Gene of Interest

Genomic library: Collections of recombinant DNA that contain pieces of the genome DNA probe: Radioactively (or fluorescently) labeled gene fragments
Green mice

cDNA library: Genomic library of protein encoding genes produced by extracting mRNA and using reverse transcriptase to make DNA
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Applications of Recombinant DNA

Recombinant DNA is used to:
Study the biochemical properties or genetic pathways of that protein Mass-produce proteins (e.g., insulin)

Sometimes conventional methods are still the better choice because of economics Textile industry can produce indigo dye in E. coli by genetically modifying genes of the glucose pathway and introducing genes from another bacterial species
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Transgenic Animals
An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal Transgenic sheep, cows, and goats have all expressed human genes in their milk,
Clotting factors Clot busters Collagen Antibodies

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Several techniques are used to insert DNA into cells to create transgenic animals
Chemicals that open transient holes in plasma membrane Liposomes that carry DNA into cells Electroporation: A brief jolt of electricity to open membrane Microinjection: Uses microscopic needles Particle bombardment: a gun like device shoots metal particles coated with foreign DNA
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Transgenic Animals
Finally, an organism must be regenerated from the altered cell If the trait is dominant, the transgenic animal must express it in the appropriate tissue at the right time in development If the trait is recessive, crosses between heterozygotes may be necessary to yield homozygotes that express the trait
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Animal Models
Transgenic animals are far more useful as models of human diseases
Example: Inserting the mutant human beta globin gene that causes sickle-cell anemia into mice

Drug candidates can be tested on these animal models before testing on humans
Will be abandoned if they cause significant side effects
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Animal Models
Transgenic animal models have limitations
Researchers cannot control where a transgene inserts, and how many copies do so The level of gene expression necessary for a phenotype may differ in the model and humans Animal models may not mimic the human condition exactly because of differences in development or symptoms
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Bioremediation
Transgenic organisms can provide processes as well as products Bioremediation: The use of bacteria or plants to detoxify environmental pollutants Examples
Nickel-contaminated soils Mercury-tainted soils Trinitrotoluene (TNT) in land mines
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Monitoring Gene Function

Gene expression DNA microarrays (gene chips) are devices that detect and display the mRNAs in a cell A microarray is a piece of glass or plastic that is about 1.5 centimeters square Many small pieces of DNA of known sequence are attached to one surface, in a grid pattern In many applications, a sample from an abnormal situation is compared to a normal control
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Monitoring Gene Function

Messenger RNAs are extracted from the samples and cDNAs are made These are differentially-labeled and then applied to the microarray The pattern and color intensities of the spots indicate which genes are expressed A laser scanner detects and computer algorithms interpret the results
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Silencing DNA
In some situations, silencing gene expression may be useful
Blocking transcription of oncogenes

Three techniques can be used to control gene expression

RNA interference Antisense sequences Knockouts from gene targeting
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Knockouts from Gene Targeting

Gene targeting is a technique that uses homologous recombination to replace a normal DNA sequence with one that cannot be transcribed or translated
This silences gene expression by creating a knockout gene Moreover, observing what happens (or not) can reveal the genes normal function

A variation of the technique exchanges genes that have an altered function, producing a knockin
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Gene Therapy
Altering genes theoretically can provide a longerlasting effect than treating symptoms The first efforts focused on inherited disorders with a known mechanism, even though the conditions are rare Gene therapy now is targeting more common illnesses, such as heart disease and cancers
Germline gene therapy - Gamete or zygote alteration; heritable; not done in humans; creates transgenic organisms Somatic gene therapy - Corrects only the cells that a disease affects; not heritable
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Assisted Reproductive Technologies

ARTs are methods that replace the source of a male or female gamete, aid fertilization or provide a uterus Developed to treat infertility but are becoming part of genetic screening

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Infertility and Subfertility

Infertility is the inability to conceive a child after a year of frequent intercourse without contraceptives Subfertility distinguishes couples who can conceive, but require longer time than usual Affect one in six couples A physical cause can be identified in 90% of cases: 30% in males, 60% in females
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Male Infertility
One in 25 men are infertile Easier to detect, but often harder to treat than female infertility Most cases of male infertility are genetic Causes of infertility include:
Low sperm count (oligospermia) A malfunctioning immune system A varicose vein in the scrotum Structural sperm defects
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Female Infertility
Many women with subfertility or infertility have irregular menstrual cycles
This makes it difficult to pinpoint when conception is most likely

Tracking ovulation cycles aids in determination of the most likely days for conception Abnormalities in any part of the female reproductive system can cause infertility
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Female Infertility
Fertility drugs stimulate ovulation but may induce release of multiple oocytes Blocked fallopian tubes can result in ectopic pregnancy (tubal pregnancy). Excess tissue growth in uterine lining may make it inhospitable for an embryo
Fibroids: benign tumors Endometriosis: buildup of uterine lining
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Assisted Reproductive Technologies (ARTs)

Examples - Intrauterine insemination - Surrogate motherhood - In vitro fertilization (IVF) - Gamete intrafallopian transfer (GIFT) - Zygote intrafallopian transfer (ZIFT) - Oocyte banking and donation - Preimplantation genetic diagnosis
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Intrauterine Insemination
Donated sperm is placed in a womans reproductive tract, typically at the cervix or in uterus Success rate is 5-15% 1790: first reported pregnancy from artificial insemination 1953: methods for freezing and storing sperm were developed Sperm catalogs list personal characteristics
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Surrogate Motherhood
In surrogate motherhood, a woman carries a pregnancy to term for another woman who cannot conceive and/or carry the pregnancy Custody rights are given up at birth A surrogate mother may or may not have contributed an oocyte Complex legal and emotional issues must be considered
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In vitro Fertilization (IVF)

For in vitro fertilization, a sperm fertilizes an oocyte in a culture dish Embryos are transferred to the oocyte donors uterus (or a surrogates uterus) for implantation 1978: First IVF child born (Louise Joy Brown) - Since then, 4 million IVF children Intracytoplasmic sperm injection (ICSI) is more effective than IVF alone

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Intracytoplasmic Sperm Injection

For cases in which sperm cannot penetrate the oocyte, IVF can be accompanied by ICSI which injects sperm directly into the oocyte ICSI allows conception in cases of low sperm count, abnormal sperm shape, sperm motility problems

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Gamete Intrafallopian Transfer (GIFT)

GIFT is a method in which superovulated oocytes from a woman and sperm from her partner are placed together in her uterine (fallopian) tube Fertilization occurs in the womans body Allows conception in cases of fallopian tube blockage 22% success rate and costs less than IVF
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Zygote Intrafallopian Transfer (ZIFT)

IVF ovum is introduced into the uterine tube and allowed to move to the uterus for implantation Also about 22% successful GIFT and ZIFT are done much less frequently than IVF - They often will not work for women with scarred uterine tubes
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Oocyte Banking and Donation

Oocytes, like sperm, can be stored frozen Only 3% successful New technique can freeze strips of ovarian tissue Difficulties because oocytes pause in meiosis II until fertilization occurs Women can store their own oocytes to have children later or prior to undergoing chemotherapy Donated oocytes can be used by women with infertility problems; 28-50% successful Embryo adoption is a variation on oocyte donation
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Preimplantation Genetic Diagnosis (PGD)

This PGD technique allows detection of genetic and chromosomal abnormalities prior to implantation One cell or blastomere of an 8-celled embryo can be removed for testing - The remaining cells will complete normal development About 29% success rate
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Extra Embryos
Sometimes ARTs leave extra oocytes, fertilized ova, or very early embryos

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Extra Embryos
A similar case to the Lyons involved a California woman named Nadya Suleman - She had eight fertilized ova left over after using six to produce her six young children - She did not want to destroy these ova or continue to store them - She was implanted with them, and in early 2009 gave birth to octuplets!
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Polar Body Biopsy

An experimental ART that increases the success of IVF Based on Mendels first law (segregation of alleles) Enables physicians to perform genetic tests on polar bodies and infer the genotype of the accompanying oocyte Oocytes that pass this test can be fertilized in vitro and the resulting embryo implanted
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Assisted Reproductive Disasters

ARTs introduce ownership and parentage issues Another controversy is that human genome information is providing more traits to track and perhaps control in coming generations So, who will decide which traits are worth living with, and which are not?
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