Transgenic Animals

Question 1

What are the genetic hybrids?

Answer 1

Mule

Liger

Tiglon/tigon

Zonkey

Zedonk

Question 2

What is a zedonk?

Answer 2

A male donkey and female zebra

Question 3

What is a zonkey?

Answer 3

A male Zebra and female donkey

Question 4

What is a tiglon/tigon?

Answer 4

A hybrid cross between a male tiger (Panthers Tigris) and a lioness (Panthera Leo)

Question 5

What is a Liger?

Answer 5

A hybrid cross between a male lion (Panthera Leo) and a females tiger (Panthera Tigris)

Question 6

What is a mule?

Answer 6

A cross between a male donkey and a female horse

Question 7

What are transgenic animals?

Answer 7

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

The use of such animals in research, medical fields, and industry has many possibilities

-However, there remain many ethical and legal questions regarding their creation and usage

Question 8

Give a Brief historical background of transgenic animals

Answer 8

In 1977 Gurdon and others showed that Xenopus oocytes provided an important expression system for molecular biology

• The genes they injected into Xenopus oocytes were transcribed and translated
• The term transgenic animals was coined by Gurdon and Ruddle in 1981

Question 9

What are the general benefits of transgenic animals?

Answer 9

• agriculture
• medicine
• industry

Question 10

What are the transgenic animal benefits to agriculture ?

Answer 10

To improve the productivity of livestock (muscle mass & wool quality)

• To improve disease resistance in animals
• Offers farmers an easy way to increase yields

Question 11

What are the benefits of transgenic animals to the medical industry

Answer 11

A. Xenopus transplantation -transplant organs may soon come from transgenic animals

B. Nutritional supplements and Biopharmaceuticals

• Products such as insulin, growth hormone, and blood anti-clotting factors have already been obtained from the milk of transgenic cows, sheep or goats
• The first transgenic cow (Rosie) produced human protein-enriched milk at 2.4 grams per liter

C. Human gene therapy
-human gene therapy involves adding a normal copy of a gene (trans gene) to the genome of a person carrying defective copies of the gene

D. Medical information
-provide fundamental inform on living organisms and human diseases

Question 12

What are the industrial applications in transgenic animals

Answer 12

Toxicity-sensitivity transgenic animals have been produced for chemical safety testing

Animals have been used as “Bioreactors” to produce proteins. Genes for desired are introduced via transgenics to the target cells

Bio steel is an extraordinary new product that may soon be used in bullet proof vests and in suture silk for stitching wounds

Question 13

What are the transgenic models in drug development ?

Answer 13

• Search for Jew drug targets
• Models for Target Validation: Desire Models, Reporter mice used to monitor gene expression, Mice with human drug targets (e.g., knock-in mice)
• Safety testing
• Protein production
• Drug screening
  
  • used for screening of many pharmaceutical drugs

Question 14

What are the problems associated with transgenic animals?

Answer 14

• Inserted gene has multiple functions
• Breeding problems
• Sometimes leads to mutagenesis and functional disorders
• Low survival rate of transgenic animals

Question 15

What are the environmental and ecological concerns of transgenic animals

Answer 15

• Competition with wild populations
• Gene introversion effects on wild gene pool
• Ecological disruption due to changes in prey size and other niche requirements
• Engineering approaches are unproven
• Bio-containment issues still not resolved

Question 16

What are the methods of producing Transgenic animals?

Answer 16

• embryonic stem cell method
• the pro nucleus method
• retrovirus mediated gene transfer

Question 17

Describe the Embryonic Stem Method

Answer 17

-Embryonic stem (ES) cells are collected from inner mass of blastocysts (about a wk old embryo)

They are then mixed with DNA containing gene of interest

• Some of the ES cells will absorb the DNA and become transformed
• Transformed ES cells are injected into inner cell mass of a host blastocyst
• Blastocyst is then injected into a foster mother
• Foster mother will give birth to baby mice

Question 18

Describe the pro nucleus method of forming transgenic animal

Answer 18

• Based on the introduction of linear DNA sequences into the chromosomes of fertilized eggs
• First, DNA with the trans gene of interest is formed.
• then there is micro injection of desired DNA into the male pro nucleus
• Leading to formation of diploid zygote
• Mitotic division & formation of two-cell embryo
• Transferring to foster mother (Pseudopregnant mother)
• Formation of new individual (Baby mice are born)

Question 19

What are the two methods of transgenic animals?

Answer 19

1. Select for stem cells expressing desired genes
   - inject transformed ES cells into inner cell mass
   - inner mass of cells from blastocyst is implanted in the uterus
2. Desired gene with vector is extracted from pronuclei of fertilized egg
   • implant in uterus

After both methods, offspring should be tested for presence of gene. Male heterozygous offspring to produce homozygous transgenic strain

Question 20

What are retrovirus mediated gene transfer?

Answer 20

Retroviruses are used as v3ctors

To transfer genetic material into the host cell, resulting in a chimera

-an organism consisting of tissues or parts of diverse genetic constitution

Question 21

What are the examples of transgenic animal?

Answer 21

Transgenic fish: Tilapia, Salmon/trout, catfish can grow up 6 times faster than wild type fish. Most have extra copies of growth hormone (GH) gene

Glowfish is a transgenic stamina of zebra fish containing red fluorescent protein gene from sea anemones

Transgenic Mice: Knick out mice

Transgenic goats(Silk-milk goats): they produce spider silk in their milk

Question 22

What are the methods of producing transgenic fish?

Answer 22

DNA is delivered to fertilized fish eggs via:

1. Micro injection
   
   • DNA injected by fine glass needle
2. Sperm mediated gene transfer
   
   • sperm coated with recombinant DNA
3. Electroporation
   
   • eggs are subject to fluctuating electric fields
4. Viral mediated gene transfer
   
   • Virus particle delivers recombinant DNA

Question 23

What are the current limitations of transgenic animals?

Answer 23

• low germline transmission
• variable number of copies inserted
• Insertion site different for each individual (pre-founder)
• uncontrolled transgene expression
• Difficulty in monitoring trans gene expression
• Insertion site affects other genes

Question 24

Describe Gene targeting technologies

Answer 24

Adding genes is only one side of the transgenic story. Genes can also be taken out

• Gene targeting involves gene knockout and targeted gene replacement
• Targeted knockout involves the deletion or disruption of a specific gene
• Gene targeting is a powerful technique used by geneticists to understand the function of genes

Question 25

What are knockout organisms/ knockouts?

Answer 25

Knockouts are used in learning about a gene which it has an unknown or incompletely known function - Researchers draw inferences from the difference between the knockout organism and normal individuals - This has been achieved in yeast, Drosophilia and mice

Question 26

What is the knockout mice?

Answer 26

A true-breeding mouse strain lacking the function of a gene. Because the gene has been replaced by either a null allele or a specific mutagenized allele

Question 27

What are the uses of a knockout mouse?

Answer 27

- As models for some human genetic disorders e.g. CF & Duchenne muscular dystrophy - To study genetic control of early development and behavior

Question 28

What are the two things required for knockout mice?

Answer 28

- A culture of mouse embryonic stem (ES) cell - A disrupted version of the gene of interest called target construct The target construct contains the Neo and thymidine kinase (TK) selectable markers and 6-10 kB of genomic DNA from the targeted gene(to ensure an adequate targeting efficiency) -The neo^r gene confer resistance to the antibiotic neomycin while the viral tk gene encides the enzyme, thymidine kinase

Question 29

What is a compete knockout?

Answer 29

This is an offshoot of gene knockout technology where a specific target gene is eliminated from a single organ in the body of an experimental animal, rather than the whole body, as with conventional gene knockout technology - It can be applied to eukaryotic and Prokaryotic systems and allows for more sophisticated experiments - The most commonly used technique in doing this is the Cre-Lox recombination system

Question 30

What is the Cre-Lox recombination?

Answer 30

Cre-Recombinase is a tyrosine recombinant enzyme derived from the P1 Bacteriophage - It is known to catalase the site specific recombination event between two DNA recognition sites(loxP sites) - DNA sequences found between two loxP sites are said to be “floxed” - Cre recombinase is a widely used tool in the field of molecular biology - The enzyme’s unique and specific recombination system is exploited to manipulate genes and chromosomes

Question 31

What is the neo^r?

Answer 31

This sequence provides resistance to the drug neomycin and provides a selectable marker for ES cells which have incorporated the targeting construct into their genomes (either randomly or by homologous recombination)

Question 32

What is the tk of the Gene targeting construct?

Answer 32

Encodes for the enzyme thymidine kinase that acts on the gancyclovir to produce a toxin. This provides a negative selectable marker for cells that have randomly incorporated the targeting construct into their genomes

Question 33

What is the *** of a gene targeting construct?

Answer 33

This region may contain nothing in the case of a “knockout” or may contain a modified form of the host gene (e.g. mutated form) that is being targeted

Question 34

What is the (purple block) of a gene targeting construct?

Answer 34

Sequences which are identical to sequences flanking the gene that is being targeted. These are the sites where homologous recombination into the host genome will occur (crossing over) during transfection

Question 35

How does a successful gene targeting works?

Answer 35

Host genome showing gene to be targeted for deletion Host genome showing the results of a successful gene targeting These ES cells will be resistant to neomycin and will survive in the presence of ganyvclovir

Question 36

Transfection of ES cells with a targeting construct can produce three different results....

Answer 36

....with respect to integration into the genome, each with possibilities for selection: A) NO INTEGRATION (most probable )- negatively selected by neomycin B) RANNDOM INTEGRATION(next probable)- negatively selected by gancyclovir C) INTEGRATION BY HOMOLOGOUS RECOMBINATION(least probable)- positively selected by both neomycin and gancyclovir

Question 37

Explain the significance of loxP

Answer 37

The loxP site can be inserted on either side of a piece of DNA (A) If Cre recombinase is then expressed in the cell, the loxP site will be cut and joined together, removing the piece of DNA between the two sites (B)Since the loxP site is directional, it can also used to invert pieces of DNA which are between it. Here two loxP sites which are inserted on either side of a piece of DNA in opposite directions. Once Cre is expressed, the loxP sites are both cut and the piece of DNA inverted and reattached