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Transgenic Animals

• traditionally selective breeding was used to enhance the genetic features of domesticated animals – developed pure breeding lines
• selective breeding time consuming, costly, can not introduce new genetic traits easily
• now foreign DNA carrying gene for certain trait (transgene) can be introduced into an animal – transgenesis
• transgenesis now used to – study gene expression, development, establishing animal model systems for human disease, and for using mammary gland to produce pharmaceuticals in milk (pharming)



developed and perfected in mice in early 80s – transgenic mice
• 3 methods based on how the DNA is introduced into the animal
• DNA can me introduced into mice by – retroviral vectors infecting embryonic cells, microinjection into enlarged sperm nucleus of fertilized egg, introduce genetically engineered embryonic stem cells into an early stage embryo


Retroviral Vector Method

• advantage – integrates the transgene into recipient cell’s genome
• only small pieces of DNA can be transferred


Steps to make Retroviral Vector

1. make defective retrovirus carrying transgene (can not replicate)
2. infect 8-cell stage mouse embryo (also infect with helper virus – increases quantities of vector DNA, but this helper viral DNA can be integrated and made into viruses)
3. implant modified embryo into foster mother mouse
4. mother gives birth to transgenic pups
5. matings are done to determine which pups have transgene in their germ line cells
6. transgenic line established from founder transgenic animals
• not used to create transgenic animals with a commercial end use


DNA Microinjection Method

• preferred method for producing transgenic mice
• steps
1. female mouse is stimulated to superovulate - produces 35 eggs instead of the normal 5-10
2. females are then mated and killed
3. fertilized eggs are flushed from oviducts and immediately microinjected -
 DNA is usually linear without prokaryotic vector DNA sequences
 male pronucleus is larger than the female and can be located with dissecting microscope
 male pronucleus is micromanipulated while the DNA is microinjected
4. 25-40 eggs are implanted microsurgically into a foster mother that has been made pseudopregnant (mate with vasectomized male)
5. foster mother will deliver pups from the inoculated eggs about 3 weeks after implantation
6. DNA from a small piece of the mouse’s tail can be assayed either by Southern blot hybridization or PCR for the presences of the transgene, mated to determine if the transgene is in the germline
• transgene integrates randomly into genome


Engineered Embryonic Stem Cell Method

• cells form the blastocyst stage of mouse embryo can proliferate in culture while retaining the ability to differentiate into all of the cells of the mouse after reintroducing into embryo (called pluripotent embryonic stem cells, ES cells)
• ES cells can be readily manipulated genetically before without altering pluripotency
• steps
1. Make vector to carry transgene – vector must have sequences homologous to distinct regions in the target genome (HB 1 and 2), the transgene which will confer a new function in the recipient, 2 selection markers for (positive/negative selection)
2. isolate cells from inner cell mass from mouse blastocyst stage embryo (ES cells)
3. transfect ES cells with vector carrying transgene
 transgene is integrated into specific location in genome of ES cells by homologous recombination
 integration must be designed to occur in genome where the DNA does not encode essential products and where there is not inhibition of transcription
4. carry out positive selection of cells that have taken up the recombinant vector – incubate cells in G-418 chemical which normally kills cells but cells that have taken up your plasmid live due to Neor gene (enrichment step in figure)
5. negatively select cells that have the plasmid integrated in the wrong location in the genome – incubate cells in ganciclovir which is not normally toxic to cells unless cells express the tk genes (1, 2, or both), cells with plasmid in the wrong place die
6. microinject selected cells back into blastocyst and implant into pseudopregnant, foster mothers
7. foster mother gives birth to transgenic pup – can establish lines from founder mice that carry transgene in germ line
Can use this technique to produce knock-out mice – target the transgene, usually a selectable marker gene, to integrate in the coding region of a gene in order to study the developmental and physiological consequences of inactivating a particular gene
• to date about 250 knockout mice have been created as animal models for studying human abnormalities

• for example – mouse rhodopsin knockout mouse causes degeneration of rod cells of the retina similar to what happens in humans with retinitis pigmentosa, scientists study the development and possible therapies


Cre-lox P Recombination system

• transgenic animals carry/express transgenes in every cell of their body
• sometimes it is useful to regulate the expression of transgenes in specific tissues
• can be used to inactivate or activate a gene in a specific tissue type
• uses cre gene from bacteriphage P1 – cre codes for recombinase that circularizes and recombines DNA from lox P sites (13 bp inverted repeats)
• steps


Transgenic Models of Alzheimer Disease

• Alzheimer disease is a degenerative brain disorder
• signs of disease - progressive loss of abstract thinking, memory, personality change, language disturbances, slowing of physical capabilities
• affects 1% of population 60-65 and 30% population over 80
• symptoms - neurofibrillary tangles accumulate within the cell body of neurons, senile plaques form at the ends of neurons (closely packed amyloid bodies which are protein fragments), neurons loose function and die
• APP protein gets cleaved incorrectly and generates protein fragments called amyloid β (A β ) proteins – inefficient clearance of these fragments leads to accumulation and inhibition of neuronal function
• mutations in APP gene found in families with high incidence of Alzheimer Disease
• transgeneic mouse models generated with APP gene mutation in specific places – PDAPP minigene
• aging mice with about 40 copies of PDAPP minigene displayed amyloid bodies, neuronal cell death, and memory defects


Transgenic Mice as Test Systems

• transgenic mice used to test whether specific proteins would be secreted into milk
• large quantities of cystic fibrosis transmembrane regulator protein (regulate the flow of chloride ions into/out of cells) needed to study its function and formulate new therapies
• conventional CFTR in vitro expression systems give low yield – accumulation of CFTR protein in the cell membrane is detrimental to cells
• ideal to use cells that continuously shed their cell membranes – mammary gland cells use this process to produce fat globules
• this idea was tested – full length CFTR cDNA was cloned into middle of a defective goat milk protein gene (retained goat promoter and termination sequences)
• transgenic mouse lines carrying CFTR sequence were established – transgenic females had CFTR gene bound to membranes of fat globules in its milk
• CFTR protein could be readily extracted form fat-rich fraction of milk


Cloning Livestock by Nuclear Transfer

• highly publicized sheep, Dolly, was cloned by transfer of a nucleus from a mammary udder cell of an adult organism – first demonstration of pluripotency of a nucleus from an adult differentiated cell
• since Dolly, cattle, goats, and pigs have also been cloned using the same method
• steps
1. embryonic, fetal, or adult donor cells are isolated, cultured, and genetically modified – prolonged culture is usually required to establish a cell line with the desired characteristics (induce cells into G0)
2. individual donor cells fused to an enucleated oocyte with short-duration electric pulses – pulses stimulate fusion and activate oocyte
3. fused cells are cultured to the blastocyst stage of development
4. blastocyst is transferred into uterus of a pseudopregnant female
5. Cconed animal is born – testing done to confirm the presence of transgene
• high loss of individuals before and after birth – not known why, if animal survives it is generally healthy (could have problems with telomeres)
• low efficiency – 277 enucleated ova fused to G0 sheep mammary cells, 29 transferred early stage embryos transferred to pseudopregnant female, 1 produced live lamb


Transgenic Birds

• several features about avian reproduction and development make production of bird transgenics by microinjection of DNA into fertilized eggs extremely inefficient
 several sperm penetrate ovum instead of one – impossible to select the one that will fuse with egg nucleus
 DNA injected into the cytoplasm of fertilized eggs does not integrate into genomic DNA
 avian ovum become enveloped in tough membrane, surrounded by albumin, and 2 membranes after fertilization – difficult injection
• steps
1. cells from the blastoderm stage of development are removed - at the time the shell hardens
2. cells are transfected with transgene
3. recipient embryos of freshly laid eggs are irradiated with gamma rays for 1 hour – this kills most of the recipient cells present, to improve the ratio of donor to recipient cells
4. transfected cells are implanted into subgerminal space of recipient embryos of freshly laid eggs
3. eggs hatch – chimeras usually are produced and breeding them produces transgenic lines
• transgenic chickens can be used to improve the genetic makeup of existing strains – in vivo resistance to viral and bacterial diseases, lower fat and cholesterol levels in eggs, better meat quality, and production of nutraceuticals