Upgrade to remove ads
Terms in this set (44)
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome.
The foreign gene is constructed using ....
Recombinant DNA methodology
In addition to a structural gene, the DNA usually includes other sequences to enable it
-To be incorporated into the DNA of the host and
-To be expressed correctly by the cells of the host.
an animal whose genetic composition has been altered by the addition of foreign DNA (the transgene)
General Transgenic Process:
- Cloned gene is injected into the nucleus of a fertilized egg.
- The inoculated fertilized eggs are implanted in a receptive female.
- Some offspring will carry the cloned genes (in all cells)
- Animals with the cloned gene in germ line cells can be bred to establish new genetic lines.
Benefits of Transgenic Animals
Animal model systems to study human diseases
Transgenic Mice Disease incducing Example:
can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans.
3 main methodologies for introducing transgene:
-engineered embryonic stem cells
Retroviral Vector Method
Cleavage-stage embryos usually at the eight-cell stage, are infected with a defective retrovirus carrying a transgene.
Implanted females (foster mothers) give birth to transgenic pups.
ADV: An effective means to integrate certain genes
DISADV; <8kb pieces of DNA
may lack essential regulatory sequences
Retroviral contamination: the helper strain retrovirus (needed to produce large amounts of vector DNA) may also be incorporated into genome
incorporation into genome is usually random
DNA Microinjection Method (1)
Eggs are obtained from donor females that have been induced (pregnant mare's serum and human gonadotrophin hormone) to superovulate.
Superovulated female produces ~35 eggs (normally 5-10)
Then mated with males, killed, fertilized eggs were flushed.
Microinjection was performed in fertilized eggs.
Purified samples of the transgene construct are microinjected into the enlarged male pronucleus of a fertilized egg.
after the female nucleus completes meiotic division to become a female pronucleus, then nuclear fusion (karyogamy) occurs.
Implanted females (foster mothers) give birth to transgenic pups
DNA Microinjection Method
several hundred pronuclei can be injected in one day
25-40 eggs are implanted in a foster mother
foster mother is made pseudopregnant through mating with a vasectomized male
gestation = 3 weeks
To identify transgenic animals:
blood from pups (tail clipping)
isolate genomic DNA
assay by Southern Blot or PCR for presence of transgene
Subsequent breeding homozygous transgenic lines
Problems with DNA Microinjection
no step is 100% efficient
~3-5% of inoculated eggs develop into live transgenic animals
66% of fertilized eggs survive injection procedure
25% of implanted eggs develop into pups
25% of the pups are transgenic
(100% x .66 x .25 x 25 = 4.125%)
not all transgenic animals are created equal!
wrong site of integration - may not be expressed
up/downstream from genes that negatively regulate transgene
copy number may be too high
may be over-expressed and disrupt normal physiology of mouse
may insert into a site that disrupts a critical gene for normal phenotype
Engineered ES Cell Method
An ES cell culture is initiated from the inner cell mass of a mouse blastocyst.
The ES cells are transfected with a transgene.
After growth, the transfected cells are identified by either the positive-negative selection procedure or PCR analysis.
Population of transfected cells can be cultured and inserted into blastocysts, which are then implanted into foster mothers.
Tissue-Specific Gene Expression
Standard transgenic methods:
gene expression or inactivation in every cell
Tissue-specific gene expression or inactivation
Tissue-specific promoters for expression
Expression or inactivation
What is cre-loxP?
Cre-Lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase.
This technology is often used in the generation of knockout and conditional knockout animals.
The loxP sequence originally comes from the P1 bacteriophage, which is a bacterial virus.
There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.
13bp 8bp 13bp
ATAACTTCGTATA - GCATACAT -TATACGAAGTTAT
Regulating a Gene Using the Cre/loxP System
The gene lacZ has LoxP sequences containing a stop signal that prevent the gene from being expressed.
When exposed to the Cre protein the LoxP and stop signal are excised and the gene is expressed.
Conditions in which the cre is present thus regulated the expression of the lacZ gene.
The Cre/lox System in Action
Two independent transgenic lines of cre and loxp were generated.
Transgenic mice containing a gene surrounded by loxP sites are mated with transgenic mice that have the cre gene expressing only in one cell type.
The resulting mice with both the cre gene and the loxP-flanked gene.
Cre-loxP system for Inactivating a gene in a specific cell type
The cre gene is placed under the control of a cell-specific promoter (pcs).
A loxP site is cloned on either side of an exon. The construct with the loxP sites is introduced into a chromosomal sire of ES cells by homologous recombination, and a transgenic mouse line is established with these cells.
In cells where both constructs are present, the Cre recombinase (red circle) is synthesized, and two Cre molecules bind to each loxP site (dotted arrow).
The loxP sites undergo recombination (black bar), leading to the excision and circularization of a loxP site and an exon (Exon 2) that is eventually degraded and the formation of an inactivated gene that is retained in the chromosome.
Cre-loxP system for Activating a transgene in a specified cell type.
The cre gene is placed as discussed previously.
A piece of DNA with loxP sites that flank a transcription termination sequence (hatched box) is cloned between a promoter (p)....Exon 1.
Homologous recombination, and a transgenic mouse line is established with these cells. In cells where both constructs are present, the Cre recombinase (red circle) is synthesized, and two Cre molecules bind to each loxP site (dotted arrow).
The loxP sites undergo recombination (black bar), leading to the excision and circularization of a loxP site and a transcription termination sequence that is eventually degraded and the formation of a transcriptionally active transgene that is retained in the chromosome.
Transgenesis with High-Capacity Vectors
large genes cannot be incorporated in regular vectors
cDNAs can be used, but generally are poorly expressed in mammalian cells.
For large gene (100kb-1000+kb) expression, high capacity vectors such as bacterial, P1 bacteriophage-derived and yeast artificial chromosomes such as YACs, BACs, MACs etc. are used.
High-Capacity Vectors - Example
Transgenic mice expressing human antibodies
MoAbs to treat cancer and other diseases
rodent MoAbs are immunogenic
hard to routinely create human MoAbs
"humanized" MoAbs - only contain 5-10% mouse content
would have to humanize for each desired MoAb
Solution: create a transgenic mouse that is capable of synthesizing an extensive array of different human antibodies
Other Applications for Transgenic Mice
-model for human genetic diseases
-model for the role of individual proteins or groups of proteins
Transgenic Mice: Degenreative Disease Applications
Alzheimer disease, Senile Dementia of the Alzheimer Type (SDAT) is the most common form of incurable, degenerative, and terminal disease.
Schematic representation of a neuron of the human cerebral cortex, showing some of the histopathological features of Alzheimer disease.
Senile plaques containing amyloid deposits and apparent cellular debris accumulate at the synaptic junction of a neuron.
Within the cell body of a neuron, neurofibrillary tangles contain aggregated cytoskeletal and other proteins.
Senile plaques are extracellular deposits of amyloid in the gray matter of the brain and are associated with degenerative neural structures and an abundance of microglia and astrocytes.
fibrillar structure = amyloid body
Aβ = principal protein of AD amyloid bodies = 4kDa, with several variants (39-42 aa)
made from APP gene (amyloid precursor protein)
Families with high incidence of AD have mutations in the APP gene
Generally difficult to study the onset of AD in humans so create a transgenic animal model!
ApoE4, the presenilin 1 gene (PS1), and the PS2 are the genes responsible for AD and can also be used to generate transgenic mice.
Conditional Regulation of Gene Expression
Tetracycline Controlled Transcriptional Activation is a method of inducible expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (etc. doxycycline).
The Tet-off system for controlling expression of genes of interest in mammalian cells.
Tet ooff system; in which DOX must be absent for transcription of the gene.
The Tet-on system works in the opposite fashion.
a protein that binds tetO promoter and turns on transgene
a tetracycline derivative, can be added to the animal's water.
Tet on system;
in which DOX must be present for transcription of the gene.
GENE UNDER THE CONTROL OF CELL SPECIFIC PROMOTER
Conditional Regulation of Gene Expression (1)
Progressive, usually apparent at 45 years of age
Loss of muscle coordination
Severe psychiatric conditions
Loss of cognitive function
Due to repeating CAG codons (glutamine) in HD gene
Repeats in exon 1
38 or more CAG codons = symptomatic
Conditional Regulation of Gene Expression (2)
Mouse Model of Huntington Disease
Variant HD gene (exon 1 with 94 CAG repeats)
tTA gene under control of promoter active in forebrain
Dox added to water during gestation
Prevents embryonic lethality
Dox removed from water after birth
Mutant HD gene expressed
Mouse version of Huntington Disease develops over time
Symptoms disappeared when Dox added back to water
New therapeutic strategies
Block mutant HD gene expression or clear neurons of mutant protein
Genetically Engineered Cell Death
Diphtheria toxin is toxic to the cells but mouse cells have no receptor.
HB-EGFr molecules are the receptor for Diphtheria toxin.
Cell membrane-localized HB-EGFr molecules (brown rectangles) are synthesized in liver cells from an HB-EGFr cDNA transgene under the control of a liver cell-specific promoter (pliver).
B. Diphtheria toxin (yellow and blue ovals) binds to an HB-EGFr and is taken into the cell. A diphtheria toxin subunit (yellow oval) is released from an endosome and inactivates EF-2. Cell death follows the cessation of protein synthesis.
Cloning Sheep Using the Nuclear Transfer Method
The nucleus of an ovum is removed (dashed arrow) with a pipette.
Cells from the mammary epithelium of an adult are grown in culture, and the G0 state is induced by inhibiting cell growth.
A G0 cell and an enucleated ovum are fused, and the renucleated ovum is grown in culture or in ligated oviducts until an early embryonic stage.
Then, implanted into a foster mother, where development proceeds to term.
Cells from blastoderm donors are removed, transfected with a transgene, and inserted into the subgerminal space of an irradiated recipient blastoderm.
Some of the resulting chickens may be chimeric.
Transgenic Sheep and Goats
July 2000, success at inserting a human transgene into a sheep for alpha1-antitrypsin, and two of the animals expressed large quantities (650 µg/ml; 50 times higher) of the human protein in their milk.
GTC Biotherapeutics, won preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the milk of transgenic goats.
Utilizing the Mammary Gland to Produce Proteins in Milk
milk is renewable
can be collected w/o harming animal
novel drug is confined to a particular compartment (mammary gland)
minimize effects on animal
normal post-translational processing of protein
purification from milk is relatively straightforward
only a small number of different proteins
Development of Transgenic Cattle
Modified from mouse transgenesis DNA microinjection protocol
poor yield is likely due to low probability of integration of the transgene
Can screen a few cells from developing blastocyst using PCR
implant only those embryos that express the transgene
Steps in the Development of Transgenic Cattle
Collection of oocytes -> in vitro maturation ->in vivo fertilization ->centrifugation of eggs -> DNA microinjection into male pronuclei -> In vitro embryonic development to blastocyte stage -> embyro implantation -> screening of offspring for transgene
Some exogenous proteins that have been expressed in mammary glands of transgenic animals
Insulin, hemoglobin, fibrinogen, growth hormone, monoclonal antibodies
Annie, is a clone of a pure bred Jersey calf whose cells were modified with genes for producing lysostaphin, a protein that kills Staphylococcus aureus bacteria, a leading cause of mastitis disease in dairy cows. She is the first transgenic cow clone engineered to resist mastitis.
These three genetically engineered and cloned pigs are considered a first step toward growing animals for use in human organ transplants.
Freeze resistance and cold tolerance
Improved product for the consumer
Potential uses of transgenic animals for pharmaceutical production
chicken , rabbit, goat, sheep
Applications of xenotransplantation
organ failure, acute liver failure, diabetes, Parkinson's disease, huntingtons disease, focal epilepsy, stroke, burn , skin injury
Sets with similar terms
Molecular Biotechnology - Exam 1
Chapter 15-16 AP Biology Test
18 Regulation of Gene Expression
Other sets by this creator
Molec Biotech Lee
Other Quizlet sets
Psych Exam 4
Middle Ages / Feudalism Study Guide
Bible: Honering our Parents