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Chapter 16Lecture and

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In 2002, this kitten named “Carbon Copy” was produced by reproductive cloning – the first cloned pet

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Chapter 16

Genetic Technology

Gene Cloning

Genomics: Techniques for Studying Genomes

Biotechnology

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Chapter Outline:

Recombinant DNA technology Use of laboratory techniques to isolate and

manipulate fragments of DNA

Recombinant DNA contains DNA from two or more sources

Once inside a host cell, recombinant molecules are replicated to produce identical copies or clones

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Gene Cloning

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Gene cloning

Creates many copies of a particular gene

To make large amounts of a specific gene’s DNA For DNA sequencing To probe for a similar gene in another species

Or to produce the protein encoded by the gene Purify protein to study structure and function Introduce into bacteria or livestock for pharmaceuticals Introduce into plants and animals to change traits Use for gene therapy to treat disease

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BIOLOGY PRINCIPLE

Biology is an experimental science

The technique of gene cloning allows researchers to study genes and gene products in greater detail.

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Gene Cloning – Step 1

Vector DNA is a carrier for the DNA segment to be cloned

When a vector is introduced into a living cell,it can replicate, making many copies

Common vectors are plasmid or viral vectors

Also need the gene of interest from some organism’s chromosomal DNA

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To insert chromosomal DNA into vector...

Cut DNA using restriction enzymes Also called restriction endonucleases Made by bacteria as protection against bacteriophages Cut at specific known restriction sites Most restriction sites palindromic May produce sticky ends

DNA ligase must be used to permanently link DNA

Creates a recombinant vector with gene inserted Will also get empty recircularized vector without insert

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Goal is to have recombinant vector taken up by bacteria Some will take up a single plasmid Most cells fail to take up a plasmid at all

Vector carries a selectable marker Adding antibiotics selects for cells expressing

antibiotic resistance gene, ampR

Gene ampR codes for beta-lactamase that degrades ampicillin, which normally kills bacteria

Growth on ampicillin plates indicates that bacteria contain plasmid with the selectable marker

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Only the cells with the plasmid grow on plates treated with ampicillin

To eliminate recircularized empty vectors, lacZ geneis built into vector Insertion of cloned DNA disrupts lacZ gene lacZ codes for beta-galactosidase, which cleaves

colorless X-Gal into a blue dye Recircularized plasmids will form blue colonies The desired recombinant vectors will form white colonies

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DNA library

Treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments

DNA library – collection of many recombinant vectors each with a fragment of chromosomal DNA

Two types of common DNA libraries Genomic library – inserts derived from chromosomal DNA cDNA library – uses reverse transcriptase to make

complementary DNA (cDNA) from mRNA Lacks introns, so simpler to use

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Electrophoresis

Technique used to separate macromolecules on a gel Can be used to separate DNA or proteins

Can be used to separate molecules based on their charge, size/length, and mass

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Polymerase chain reaction (PCR)

Allows us to amplify DNA (make many copies) No vectors or host cells needed

Ingredients: Primers that match sequences at each end of the DNA fragment dNTPs (deoxynucleotide triphosphates) Taq polymerase – a heat-stable form of DNA polymerase called

DNA run through repeated cycles of denaturation, annealing and synthesis Thermocycler machine automates this process

After 30 cycles of amplification, a DNA sample will increase 230-fold

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Genomics – the molecular analysis of the entire genome (all the DNA) of a species

Two phases Mapping of genome Functional genomics

How do specific DNA sequences impact structure and function of cells and the organism?

Expression analysis – which genes turn on or off in particular cells

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Genomics: Techniques for Studying Genomes

DNA sequencing

Determines base sequence of DNA

Dideoxy chain-termination method Dideoxynucleoside triphosphates (ddNTPs) are missing

the 3’–OH group and cause chain to terminate 4 tubes with copies of the single stranded DNA, each with

a different labelled nucleotide DNA polymerase will make complementary strand until

ddNTP inserted and chain terminates After electrophoresis, DNA sequence can be read by

determining which base is at the end of the DNA strand

Procedure has been automated using fluorescently labeled ddNTPs all in one tube 26

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3′

Recombinant vector

Annealingsite

5′

Primer

Many copies of the recombinant vector, primer,dNTPs, fluorescently labeled dideoxynucleotides(ddNTPs), and DNA polymerase are mixedtogether. Incubate to allow the synthesis of DNA.

CACCGTAAGGACTddGCACCGTAAGGACddTCACCGTAAGGAddCCACCGTAAGGddACACCGTAAGddGCACCGTAAddGCACCGTAddACACCGTddACACCGddTCACCddGCACddCCAddCCddAddC

Separate newly made strands bygel electrophoresis.

981 2 3 4 5 6 7

1011121314

GT G G C A T T C

C

TG

AC

Sequence to be analyzed(target DNA)

Synthesis of short DNA strandsuntil a ddNTP is added.

GTCAGGAATGCCAC

Sequencededucedfrom gel

(a) Dideoxy DNA sequencing method

Fluorescencedetector

C A C C G T A A G G A C T G

(b) Output from automated sequencing

Longest DNAstrand

Laserbeam

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A microarray can identify which genes are transcribed by a cell

DNA microarray or gene chip

Monitors expression of thousands of genes simultaneously

Short sequences of known genes attached to spots on slide

Goal is to find out which genes are transcribed into mRNAin a particular sample of cells mRNA from those cells is used to make fluorescently labeled cDNA

cDNAs that are complementary to the DNAs in the microarray will hybridize

High fluorescence intensity in a spot indicates high levels of cDNA in the sample

Technologies that involve the use of living organisms to benefit humans

12,000 years ago – domestication of livestock Now – molecular genetics

Important medicines are now produced by recombinant organisms Tissue plasminogen activator – for blood clots Factor VIII – for hemophilia Insulin – for diabetes

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Biotechnology

Insulin

In 1982, the U.S. FDA approved sale of human insulin made by recombinant bacteria

Prior to 1982, insulin was isolated from cattle Some people developed allergies and had to use cadaver insulin

Insulin composed of two polypeptides – A and B A and B coding sequence inserted into E.coli Fusion proteins extracted and β-galactosidase removed Purified A and B chain mixed to form functional protein

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BIOLOGY PRINCIPLE

Biology affects our society

This method of making insulin, which was developed by researchers, is now used to make insulin for most

people with insulin-dependent diabetes.

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Gene replacement and knockout

Used to understand gene function and human disease

A transgenic organism carries genes introduced using molecular techniques Also called Genetically Modified Organism (GMO)

Gene replacement – cloned gene recombines with normal gene on a chromosome Only 1 of 2 copies replaced, creating heterozygote Heterozygotes can be crossed to yield homozygotes

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Gene knockout – if cloned gene is a mutation that inactivates function, homozygote will not have gene function

Very useful for studying human disease Used to create models to study disease Used to test effect of therapies

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Molecular pharming

Production of medically important proteins in livestock mammary glands

Certain proteins are more likely to function when expressed in mammals Post-translational modification Degraded or improperly folded in bacteria High yield in cows

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Strategy: Clone gene next to the promoter of a gene specifically expressed in mammary cells

Vector injected into oocyte, fertilized and implanted

Protein is purified out of milk

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Transgenic plants

Somewhat easier because plant cells are totipotent

Cloned genes can be introduced into somatic tissue and entire plant regenerated using hormones

Agrobacterium tumefaciens naturally infects plant cells and causes tumors Contains Ti plasmid that integrates into host chromosome Codes for plant growth hormones that form crown gall tumor

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Transgenic plants

Ti plasmid modified to introduce cloned genes KanR used as a selectable marker for kanamycin resistance Contain convenient restriction sites

Transformed cells plated on media with kanamycin (to kill nontransformed cells) and carbenicillin(to kill Agrobacterium)

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Successful example of the use of transgenic plants has involved the introduction of genes from Bacillus thuringiensis (Bt)

Bacterium produces toxins that kill certain caterpillars and beetles and has been widely used as an insecticide for several decades

Bt varieties of plants produce the toxins themselves – therefore are resistant to many caterpillars and beetles

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BIOLOGY PRINCIPLE

Biology affects our society

The field of agriculture has been greatly influenced by the development of genetically modified organisms (GMOs) such as Bt corn.

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Cloning in mammals

Identical twins are genetic clones from a single fertilized egg that split early in development

Plants can be cloned from somatic cells

For decades it was believed that mammalian somatic cells could not be used for cloning

1996, Dolly was the first cloned lamb

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Mammary cells from adult sheep removed

Fused diploid mammary cell with enucleated sheep oocyte

Zygote implanted

Dolly and donor were almost genetically identical Same set of genes Minor differences due to differences in

mitochondrial DNA and maternal effect genes

Cloning achieved now in several mammal species

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Stem cells

Have potential to treat a variety of human disorders

Stem cells are undifferentiated cells that can divide and create the bodies of plants and animals They have the capacity to divide Can differentiate into specialized cell types

Embryonic stem cells (ES cells) and embryonic germ cells (EG cells) are pluripotent They can differentiate into many different cell types Thus potential for many different diseases

Stem cells already in use in bone marrow transplants51

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Blaese and colleagues performed the first gene therapy to treat ADA deficiency

Gene therapy puts cloned genes into living cells to cure disease

Over 4,000 diseases involve mutations in single genes

Adenosine deaminase deficiency prevents proper metabolism of nucleosides

Deoxyadenosine accumulates and results in destruction of B and T cells Leads to severe combined immunodeficiency syndrome (SCID) Fatal at an early age (1 or 2 years old)

FEATURE INVESTIGATION

Three treatment approaches: Bone marrow transplant Purified ADA enzyme Gene therapy

Gene therapy September 14, 1990 Removed lymphocytes from girl Treated with retroviral vector containing ADA gene Returned cells to her bloodstream

Results suggest that this first gene therapy trial may offer benefit, but patients were also treated with ADA – so could not know for sure



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Retrovirus

HYPOTHESIS Infecting lymphocytes with a retrovirus containing the normal ADA gene will correct the inherited deficiency of the mutant ADA gene in patients with ADA deficiency.

KEY MATERIALS A retrovirus with the normal ADA gene.

Remove ADA-deficient lymphocytes from thepatient with severe combined immunodeficiencydisease (SCID).

Culture the cells in a laboratory.

Lymphocyte

Infuse the ADA-gene-corrected lymphocytes backinto the SCID patient.

LymphocytesLymphocyte

X

Mutant ADA gene

Normal ADA gene

Retroviral inserted DNA

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Experimental level Conceptual level

3 Infect the lymphocytes with a retrovirus that contains the normal ADA gene. As described in Chapter 18, retroviruses insert their DNA into the host cell chromosome as part of their reproductive cycle.

Retrovirus withADA gene



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0 365 730 1,095 1,460

Protocol day

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5 CONCLUSION The introduction of a cloned ADA gene into lymphocytes via gene therapy resulted in higher ADA function, even after 4 years.

SOURCE Blaese, R. Michael et al. 1995. T lymphocyte- directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science 270:475–480.

THE DATA

Transfusions stopped


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1 Chapter 16 Lecture and Animation Outline Copyright © McGraw-Hill Education. Permission required for reproduction or display. To run the animations you