Transgenic Animals

Question 1

What is the traditional method used for improving livestock and other domesticated animals genetically for desirable traits?

Answer 1

Selective breeding through many generations of selective matings.

Question 2

What are some traits that can be improved through traditional breeding methods?

Answer 2

Milk yield, wool characteristics, rate of weight gain, and egg-laying frequency.

Question 3

Why is it difficult to introduce new genetic traits using selective-breeding methods once an effective genetic line has been established?

Answer 3

Because a new breeding line may carry both desirable and undesirable genes, and crossing may result in diminished production levels.

Question 4

What is transgenesis?

Answer 4

Transgenesis is the process of introducing foreign DNA (transgene) into the genetic composition of an animal, resulting in a transgenic animal.

Question 5

What are some practical applications of transgenic technology?

Answer 5

Studying gene expression and development, establishing animal model systems for studying human diseases, producing foreign proteins in bird eggs, and producing pharmaceuticals in the mammary gland.

Question 6

Why is the mammary gland often used for producing pharmaceuticals through transgenic technology?

Answer 6

Milk is a renewable, secreted body fluid that can be collected frequently without harm to the animal, and purification of the protein from milk is relatively straightforward due to the small number of different proteins in milk.

Question 7

What is the disadvantage of using retroviral vectors for transgenesis?

Answer 7

Retroviral vectors are effective in integrating the transgene into the genome of a recipient cell. However, they can only transfer small pieces of DNA and may lack essential adjacent sequences for regulating gene expression

Question 8

What is a major drawback of using retroviral vectors for transgenesis?

Answer 8

The genome of the retroviral strain used to create the vector DNA can be integrated into the same nucleus as the transgene, potentially leading to retroviral contamination. Additionally, transgenes introduced on retroviral vectors may be silenced in mouse embryos.

Question 9

What are lentiviruses and why are they used for transgenesis?

Answer 9

Lentiviruses are a group of retroviruses that can deliver large segments of DNA into the host genome, are stable for relatively long periods, have low immunogenicity, and can infect both dividing and nondividing cells. Lentiviral vectors are used for transgenesis because they are capable of delivering larger DNA fragments and do not silence transgenes in embryos.

Question 10

What are some applications of transgenic mice in biomedical research?

Answer 10

Transgenic mice have been used to study gene regulation, tumor development, immunological specificity, molecular genetics of development, and many other biological processes. They have also been used as biomedical models for various human genetic diseases and to examine the feasibility of industrial production of human therapeutic drugs by domesticated animals.

Question 11

What are the three methods for introducing DNA into mice for transgenesis?

Answer 11

The three methods for introducing DNA into mice for transgenesis are:

1. Retroviral vectors that infect the cells of an early-stage embryo prior to implantation into a receptive female.
2. Microinjection into the male pronucleus of a fertilized egg.
3. Introduction of genetically engineered embryonic stem cells into an early-stage developing embryo before implantation into a receptive female.

Question 12

What are some advantages of lentiviral vectors for transgenesis?

Answer 12

Lentiviral vectors have several advantages for transgenesis, including the ability to deliver larger DNA fragments, stability for long periods, low immunogenicity, and the ability to infect both dividing and nondividing cells. They are also not silenced in embryos and do not carry the risk of retroviral contamination.

Question 13

What is the preferred method for producing transgenic mice and why?

Answer 13

The preferred method for producing transgenic mice is microinjection of DNA into the male pronucleus of fertilized eggs. This method is preferred because it allows for a higher number of available fertilized eggs by stimulating donor females to superovulate, resulting in a larger number of eggs for injection. Additionally, the injected DNA integrates at random sites within the genome, allowing for the creation of transgenic lines of mice carrying functional transgenes.

Question 14

How is the superovulation of female mice achieved in the microinjection method?

Answer 14

Superovulation of female mice is achieved by giving them an initial injection of pregnant mare’s serum and another injection of human chorionic gonadotropin about 48 hours later. This stimulation results in the production of approximately 35 eggs per superovulated mouse, as opposed to the normal 5 to 10 eggs.

Question 15

What happens after the fertilized eggs are collected for microinjection?

Answer 15

After the fertilized eggs are collected from the oviducts of superovulated female mice, microinjection of the DNA construct usually occurs immediately. The male pronucleus, which can be located using a dissecting microscope, is injected with the transgene construct, which is often in a linear form and free of prokaryotic vector DNA sequences.

Question 16

How are the implanted eggs in the foster mother prepared for implantation in the microinjection method?

Answer 16

The foster mother, which has been made pseudopregnant by being mated to a vasectomized male, receives 25 to 40 implanted eggs microsurgically. Copulation is the only known way to prepare the uterus for implantation in mice, and in this case, none of the eggs from the foster mother are fertilized due to the vasectomized mate. The foster mother then delivers pups from the inoculated eggs about 3 weeks after implantation.

Question 17

How are transgenic animals identified in the microinjection method?

Answer 17

transgenic animals can be identified by assaying DNA from a small piece of the tail using Southern blot hybridization or polymerase chain reaction (PCR) for the presence of the transgene. A transgenic mouse can also be mated to another mouse to determine if the transgene is in the germ line of the founder animal. Subsequently, progeny can be bred with each other to form pure (homozygous) transgenic lines.

Question 18

What are pluripotent embryonic stem cells?

Answer 18

Pluripotent embryonic stem cells are cells from the blastocyst stage of a developing embryo that can proliferate in cell culture and retain the capability to differentiate into all other cell types, including germ line cells.

Question 19

How can embryonic stem cells be genetically engineered without altering their pluripotency?

Answer 19

Embryonic stem cells can be genetically engineered by transfection with a DNA vector that is designed to integrate within a specific chromosomal location. This allows for the integration of a functional transgene at a specific site in the genome without affecting pluripotency.

Question 20

What is the advantage of using the Engineered Embryonic Stem Cell Method over other methods of genetic engineering?

Answer 20

The Engineered Embryonic Stem Cell Method allows for precise genetic engineering without the randomness of integration that is inherent in DNA microinjection and retroviral vector systems. It also avoids interference with essential developmental or cellular functions, as the transgene is integrated into a non-essential region of the genome.

Question 21

What is positive-negative selection in the Engineered Embryonic Stem Cell Method?

Answer 21

Positive-negative selection is a procedure used in the Engineered Embryonic Stem Cell Method to enrich for cells with DNA integrated at the target site. It involves using a DNA vector that contains two blocks of DNA sequences homologous to the target site, as well as genes for resistance to a compound (e.g., G-418) and herpes simplex virus thymidine kinase (HSV-tk1 and HSV-tk2). Cells that have integrated the vector at the target site will be resistant to the compound and negative for HSV-tk, while cells with spurious integration will not.

Question 22

What are the considerations for selecting the target site for integration in the Engineered Embryonic Stem Cell Method?

Answer 22

The target site should be located in a section of genomic DNA that encodes no essential products so that it does not interfere with any developmental or cellular functions. It should also be in a part of the genome that allows for transcription, such as euchromatin rather than heterochromatin.

Question 23

How are transgenic animals generated using the Engineered Embryonic Stem Cell Method?

Answer 23

Transgenic animals can be generated using the Engineered Embryonic Stem Cell Method by first initiating an embryonic stem cell culture from the inner cell mass of a blastocyst. The embryonic stem cells are then transfected with a transgene using a DNA vector designed for positive-negative selection. After selection and enrichment of transfected cells, they can be inserted into blastocysts and implanted into foster mothers. Transgenic lines can be established from founder mice that carry the transgene in their germ lines.

Question 24

What is the purpose of the Cre-loxP recombination system in transgenic mice?

Answer 24

The Cre-loxP recombination system is used to selectively regulate the expression of a gene within a specific somatic tissue or cell type in transgenic mice.

Question 25

What is bacteriophage P1 and how is it involved in the Cre-loxP recombination system?

Answer 25

Bacteriophage P1 is a type of virus that infects Escherichia coli. It has a double-stranded DNA genome and is about 100 kb in length. The circularized P1 genome, which is mediated by the product of the cre gene (Cre recombinase protein), is used as a template for replication or integration into the E. coli chromosome. The Cre recombinase specifically cleaves and recombines the DNA of loxP sites, which are used in the Cre-loxP recombination system.

Question 26

What is a loxP site and how does it function in the Cre-loxP recombination system?

Answer 26

A loxP site is a specific DNA sequence that consists of two 13-base-pair inverted repeats separated by an 8-base-pair spacer sequence. The Cre recombinase binds to the loxP site and cleaves the DNA within the spacer region, leading to the exchange and joining of DNA strands to form recombined DNA molecules. The outcome of the recombination event depends on the orientation of the repeats of the loxP sites. If the repeats are in opposite directions, the DNA between the two loxP sites is inverted. If the repeats are in the same orientation, the intervening sequence is excised.

Question 27

How is the Cre-loxP system used to produce cell-specific gene modifications in mice?

Answer 27

The Cre-loxP system is used to produce cell-specific gene modifications in mice by first isolating the cre gene and placing it under the control of a cell-specific promoter. Transgenic mice with the cre gene construct are established, and the tissue specificity of the Cre activity is confirmed. Then, a loxP site with repeat sequences in the same direction is inserted on either side of a cloned DNA sequence, such as a cloned exon. The construct is integrated into a chromosome site of embryonic stem cells by homologous recombination. These cells are selected, cultured, and used to establish a transgenic mouse line. Finally, a transgenic mouse with the tissue-specific cre transgene is mated with a transgenic mouse with the integrated loxP-flanked sequence, and the DNA between the two loxP sites is deleted after the cre transgene is expressed, leading to cell-specific gene modifications in the resulting offspring.

Question 28

What is CRE-LOX P and what are the components involved?

Answer 28

CRE-LOX P is a genetic system that involves the Cre recombinase enzyme and lox P sequences. The lox P sequences consist of two 13-base pair repeats separated by an 8-base pair spacer. The Cre recombinase recognizes and binds to the lox P sequences, bringing them together and cutting within the spacer region.

Question 29

What happens when the lox P sequences are moving in the same direction in CRE-LOX P?

Answer 29

When the lox P sequences are moving in the same direction, the Cre recombinase enzyme cuts within the spacer region, resulting in the removal of whatever is in the spacer region.

Question 30

How can the Cre recombinase gene be cloned in an organism in CRE-LOX P?

Answer 30

The Cre recombinase gene can be cloned under the control of a cell/tissue-specific promoter in an organism, such as a hair promoter. This allows for tissue-specific expression of the Cre recombinase enzyme, making the animal transgenic.

Question 31

How can the CRE-LOX P system be used for gene inactivation?

Answer 31

To achieve gene inactivation, the gene that needs to be inactivated is placed in the spacer region between the lox P sequences, which are moving in the same direction. When the Cre recombinase is activated, it binds to the lox P sequences, brings them together, cuts in the spacer region, and ligates the DNA, resulting in the removal of the gene in the spacer region.

Question 5: What is the benefit of tissue-specific gene inactivation in CRE-LOX P?

Answer: Tissue-specific gene inactivation allows scientists to study the importance of a specific cell and its genes without affecting the other parts and functioning of the organism, preventing the destruction of the organism.

Question 6: How can the CRE-LOX P system be used for gene activation?

Answer: Gene activation can be achieved by creating a recombinant DNA construct with a cell-specific promoter that controls the expression of Cre recombinase. Another recombinant DNA construct is created with the lox P sequences, its promoter, and the gene of interest flanked by transcriptional termination sequences. When the Cre recombinase is triggered by an inducer, it recognizes and cuts out the transcriptional termination sequences, allowing for the expression of the gene of interest.

Question 7: When is gene activation triggered in CRE-LOX P?

Answer: Gene activation is triggered when the animal is at a specific stage of development where the gene can be expressed safely, usually by adding an inducer, such as a chemical agent to water or a regulatable promoter triggered at a specific stage of the animal’s development (cell-specific, physiology-specific).

Question 32

What is the benefit of tissue-specific gene inactivation in CRE-LOX P?

Answer 32

Tissue-specific gene inactivation allows scientists to study the importance of a specific cell and its genes without affecting the other parts and functioning of the organism, preventing the destruction of the organism.

Question 33

How can the CRE-LOX P system be used for gene activation?

Answer 33

Gene activation can be achieved by creating a recombinant DNA construct with a cell-specific promoter that controls the expression of Cre recombinase. Another recombinant DNA construct is created with the lox P sequences, its promoter, and the gene of interest flanked by transcriptional termination sequences. When the Cre recombinase is triggered by an inducer, it recognizes and cuts out the transcriptional termination sequences, allowing for the expression of the gene of interest.

Question 34

When is gene activation triggered in CRE-LOX P?

Answer 34

Gene activation is triggered when the animal is at a specific stage of development where the gene can be expressed safely, usually by adding an inducer, such as a chemical agent to water or a regulatable promoter triggered at a specific stage of the animal’s development (cell-specific, physiology-specific).

Question 35

What does CRISPR stand for and what is its role in bacteria’s immune system?

Answer 35

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a region of DNA in bacteria that contains short sections of DNA from invading pathogens, which are known as spacers. These spacers are used by bacteria to build their immune system against the pathogens and protect themselves from future infections.

Question 36

What is CAS in CRISPR-Cas, and what is its function?

Answer 36

CAS stands for CRISPR-Associated Protein. CAS proteins, specifically CAS9, are endonucleases that work together with CRISPR RNA to cut and excise the invading DNA from pathogens. CAS9 is guided by the CRISPR RNA to the target sequence on the invading DNA, where it introduces a double-stranded break, preventing infection.

Question 37

What is the role of PAM in the CRISPR-Cas system?

Answer 37

PAM stands for Protospacer Adjacent Motif. PAM is a specific sequence that must be present in the invading DNA for CAS9 to cut it. PAM acts as a signal for CAS9 to recognize and bind to the target sequence on the invading DNA. It ensures the specificity of the CRISPR-Cas system, as CAS9 will only cut DNA with the PAM sequence adjacent to the target sequence.

Question 38

What are the options for repairing the double-stranded break caused by CRISPR-Cas system?

Answer 38

There are two options for repairing the double-stranded break caused by the CRISPR-Cas system:

Non-homologous end joining (NHEJ): This repair mechanism is prone to errors as it can cause random insertions or deletions, leading to mutations.

Homology-directed repair (HDR): This repair mechanism uses a template DNA, such as a donor plasmid or a single-stranded oligonucleotide, to repair the cut DNA. HDR is more precise and can be used to introduce specific changes in the DNA, such as replacing a defective gene with a functional copy.

Question 39

How is the CRISPR-Cas system used in the commercial setting?

Answer 39

In the commercial setting, the CRISPR-Cas system is used for various applications, such as gene editing and gene therapy. One common approach is to use a single-guide RNA (sgRNA) molecule that is designed to bind to the target sequence in the DNA. Along with the sgRNA, the Cas9 endonuclease is introduced, either as mRNA or as a protein. The Cas9 endonuclease cuts the DNA at the target sequence guided by the sgRNA. This cut triggers the repair mechanisms in the cell, which can be either NHEJ or HDR. Additionally, a donor plasmid or a single-stranded oligonucleotide with the desired DNA sequence can be introduced along with the sgRNA and Cas9 to facilitate HDR and replace a defective gene with a functional copy.

Question 40

What are the two main methods to silence gene expression in animal cells for studying biological processes?

Answer 40

The two main methods to silence gene expression in animal cells are the knockout method and the knockdown method.

Question 41

Describe the knockout method for silencing gene expression in animal cells

Answer 41

The knockout method involves targeted disruption of a gene by homologous recombination in embryonic stem cells, resulting in the complete abolishment of gene expression.

Question 42

Describe the knockdown method for silencing gene expression in animal cells.

Answer 42

The knockdown method involves decreasing the expression of a target gene by preventing messenger RNA (mRNA) translation using RNA interference (RNAi), a natural mechanism for regulating gene expression by endogenous RNA molecules in animals and plants.

Question 43

How does RNAi work in the knockdown method of gene silencing?

Answer 43

In RNAi, double-stranded RNA is cleaved into smaller double-stranded RNA molecules called small interfering RNA (siRNA) by an enzyme called Dicer. The siRNA, along with a large nuclease complex called RNA-induced silencing complex (RISC), binds to homologous sequences on mRNA molecules and degrades them, preventing the synthesis of the encoded protein.

Question 44

How is the knockdown method implemented in transgenic mice?

Answer 44

To implement the knockdown method in transgenic mice, a small region of the target gene’s sequence is cloned into a vector as an inverted repeat separated by a spacer region. The RNA transcript produced from this sequence forms a short hairpin RNA (shRNA) due to intramolecular basepairing. The shRNA is then processed by the host cell’s Dicer and RISC proteins into siRNA, which reduces the expression of the target gene.

Question 45

What are some advantages of using the knockdown method in transgenic animals?

Answer 45

Some advantages of using the knockdown method in transgenic animals include the ability to selectively decrease the expression of a specific gene, the potential to study gene function in vivo, and the possibility of generating transgenic animals with reduced gene expression for research or practical applications in agriculture and biotechnology.

Question 46

Can the knockdown method be applied to species other than mice?

Answer 46

Yes, the knockdown method has been successfully applied to a variety of animals, including cows, pigs, goats, frogs, and rats, by introducing the transgenic construct encoding the shRNA into the embryos or cells of these animals.

Question 47

Why is it important to use shorter sequences in the knockdown method?

Answer 47

Shorter sequences are used in the knockdown method to avoid the general downregulation of translation that is often elicited with longer sequences, which is thought to occur as part of the viral defense response.

Question 48

What is the role of Dicer and RISC in the knockdown method?

Answer 48

Dicer is an enzyme that cleaves double-stranded RNA into smaller siRNA molecules, and RISC is a large nuclease complex that separates the strands of siRNA. Together, Dicer and RISC process the shRNA or siRNA molecules into active forms that bind to mRNA molecules and degrade them, leading to reduced expression of the target gene.

Question 49

What are some potential applications of the knockdown method in biotechnology?

Answer 49

Some potential applications of the knockdown method in biotechnology include the generation of transgenic animals with reduced expression of specific genes for research purposes, the development of genetically modified animals with desired traits in agriculture, and the study of gene function in vivo to understand biological processes and disease mechanisms.

Question 50

What is the main protein found in the amyloid bodies of Alzheimer disease patients?

Answer 50

The main protein found in the amyloid bodies of Alzheimer disease patients is Aβ (amyloid β, β-protein, β-amyloid protein, or β/A4). Aβ protein is derived from an internal proteolytic cleavage of the β-amyloid precursor protein (APP).

Question 51

How are mouse models used to study Alzheimer disease?

Answer 51

Mouse models for Alzheimer disease are created using transgenes that contain mutations in the APP gene that occur in some families with a high incidence of early onset of Alzheimer disease. These transgenic mice display Alzheimer disease-like features, including the formation of amyloid plaques, neuronal cell death, and memory defects, which allows researchers to study the onset and pathogenesis of Alzheimer disease in an animal model.

Question 52

What are some other mouse models for human genetic diseases?

Answer 52

• amyotrophic lateral sclerosis,
• Huntington disease, arthritis,
• muscular dystrophy,
• tumorigenesis, hypertension, -
• neurodegenerative disorders,
• endocrinological dysfunction,
• coronary disease,