Animal Transgeneis

Transgenic animals are genetically engineered animals that have a foreign gene or genes of interest introduced into their genome, which are transmitted and expressed by their progeny.

The creation of novel ways of gene delivery in mammals was aided by advances in assisted reproductive technologies (ART) and molecular biology procedures, which hastened the development of transgenic technology. The idea of genetic manipulation of animal by introducing genes into fertilized eggs became real during the 1980s. Gradually transgenesis has become a mighty technique for analyzing the function of a plethora of genes, for generating animal models of human diseases, and for producing pharmaceutically important proteins secreted through milk.

The process of expressing a gene of pharmaceutical proteins in the mammary gland is termed as “pharming.” Mammary gland is chosen for expressing the proteins because milk can be collected from the animal without any harm to the animal.

There are several steps in the process of animal transgenesis.

24.9.1 Pronuclear Microinjection

In this technique, exogenous DNA is injected into the male pronucleus of zygotes with the help of a pulled glass needles. Foreign DNA molecules become inserted into their genomes at random sites. Embryos produced are subsequently transferred into a surrogate mother where viable offspring grow.

24.9.2 Embryonic Stem Cell Engineering

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of the blastocyst. These cells have the ability to divide indefinitely under ideal culture conditions. Because of this property, ES cells can be easily propagated and manipulated by inserting a DNA construct encoding the desired gene or genes. The procedure entails isolating and cultivating ES cells in vitro prior to inserting the transgene. The transgenic ES cells are then separated from non-transgenic cells and allowed to multiply to generate transgenic ES cell colonies.

The chimeric animals are then evaluated for germline transmission, and pure transgenic animals are created using various breeding procedures. The use of ES cells for transgenesis has a number of advantages, as transgene integration can be tested using selectable markers.

24.9.3 Sperm-Mediated Transgenesis

Spermatozoa can naturally uptake exogenous DNA by using a simple incubation process. DNA attaches to the plasma membrane of sperm cells via a DNA-binding protein found in sperm. The mechanism mediated by CD4 molecules internalizes about 15-20% of the DNA attached to sperm. In order to insert the gene of interest within sperm various techniques are used to transfer sperm carrying a gene of interest into oocytes, with variable degrees of success. Electroporation or lipofection can help enhance DNA uptake by sperm. Exogenous DNA attaches to the subacrosomal region of the sperm head of different species. Exogenous DNA interacts with DNA-binding protein(s) (DBP) of 30-35 kDa on the sperm cell surface, forming a DNA/DBP complex that is triggered by CD4-mediated internalization and is dependent on MHC class II expression. DNA/DBP/CD4 enters the

Fig. 24.9 Cre-lox system of recombination. (a) Excision—cis placement of IoxP sites in same direction. (b) Inversion—cis placement of loxP sites in opposite direction. (c) Translocation—trans placement of loxP sites

nucleus and reaches the nuclear matrix. A small percentage of foreign DNA recombines with the sperm chromosomal genome at a few “accessible” chromatin locations.

24.9.4 Virus-BasedTransgenesis

Viral vectors have been used to mediate foreign gene transfer by delivering and integrating transgenes into the host genome. Viral vectors can be divided into non-integrating viral vectors (e.g., adenoviral vectors), and integrating viral vectors that are mostly derived from a retrovirus, lentivirus, and adeno-associated virus (AAV).

Viral vectors can be employed to perform transgenesis into zygotes in two ways: zona-free embryo viral transduction and perivitelline space (subzonal) injection.

24.9.5 Recombinase-Mediated Transgenesis

Recombinases are natural enzymes that facilitate genetic recombination at specified sites. Recombinases can accomplish deletions, insertions, and inversions in DNA sequences by interacting with their own recognition sites. For a variety of reasons, site-specific recombinases have been incorporated into genome editing initiatives. Cre recombinase, Flp recombinase, and PhiC31 integrase have all been used to manipulate livestock genomes.

The basis of the Cre-lox recombinase system is the ability of the P1 bacteriophage cyclization recombination (Cre) recombinase gene (cre) to effect recombination between pairs of 34 base pair loxP sites. This 34-bp sequence consists of two 13-bp inverted or palindromic repeats separated by an 8-bp spacer region (Fig. 24.9). The Flp/FRT system, which is derived from the yeast Saccharomyces cerevisiae, works similarly to the Cre/loxP system in that flippase (Flp) detects and cleaves two FRT recognition sites. The PhiC31 integrase from Streptomyces bacteriophage has been used to mediate homologous recombination between attB and corresponding pseudo attP sites.

24.9.6 Transposon-Mediated Transgenesis

DNA transposons are mobile DNA elements that can integrate into the chromosomes of host cells, allowing them to function as gene transfer vectors. These systems are used as binary tools consisting of a transposon vector and a supply of the transposase enzyme. Mechanism of transposition involves excision of a transgenic cassette of interest flanked by transposon-inverted repeat sequences from a plasmid vector, followed by genomic integration (Fig. 24.10).

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• Gene knock-in: Gene knock-in technology modifies the genetic locus of interest by replacing DNA sequence information one-for-one or by adding sequence information not found on the genetic locus.

• Gene knockout: Gene knockout is the total removal or permanent deactivation of a gene through genetic engineering.

• Gene knock down: Gene knock down is the deactivation or suppression of genes, rather than complete deletion.

• Conditional knockout: Conditional knockout is an approach to knockdown studies in genes that would be lethal if they were completely knocked out.

Fig. 24.10 The plasmid-based binary transposon system. Binary transposon platform is made up of the transposon construct harboring the gene of interest flanked by inverted terminal repeats (ITR) and the transposase protein. After the components are delivered to the cells, the transposase protein is translated and attaches to the ITRs flanking the transgene. Transposase catalyzes the transposon’s excision and subsequent genomic integration. Integrations happen at sequence motifs TA or TTAA. Duplication of these motifs occurs at the insertion site to flank the transposon

24.9.7 Application

24.9.7.1 Improving Milk Production

and Composition

The creation of transgenic animals that produce more milk, yield milk with better nutrient content, or yield milk with beneficial protein, is required to increase livestock growth or survivability through milk composition alteration. Increased expression of a number of these proteins in milk may benefit the developing offspring’s growth, development, health, and survival. Bioactive substances like insulin-like growth factor (IGF), transforming growth factor (TGF-α), and lactoferrin in the milk play important roles in the development and maturation of the gut, the immune system, and the endocrine organs of neonates. Transgenic animal-secreting proteins with physiological roles within the mammary gland itself such as lactalbumin, lysozyme, lysostaphin, or other antimicrobial peptides have been produced.

24.9.7.2 Improving Growth and Carcass Composition

Porcine growth hormone (PGH) genes were introduced into the pig genome, which boosted the growth rate without causing arthritis or aberrant skeletal growth.

Transgenic pigs with a human metallothionein promoter/porcine growth hormone gene construct showed significant improvements in economically important traits like growth rate, feed conversion, and body fat/muscle ratio while avoiding the pathological phenotype seen in previous growth hormone constructs. Transgenic pigs for human insulin-like growth factor-I produce 30% more loin mass, 10% more carcass lean tissue, and 20% less total carcass fat. The generation of the first pigs transgenic with a spinach desaturase gene, which produces greater levels of unsaturated fatty acids, was a significant step toward the production of healthier pork recently. In striated muscle, these pigs have a greater ratio of unsaturated to saturated fatty acids, indicating that they eat a diet rich in unsaturated fatty acids.

24.9.7.3 Generating Disease-Resistant Animals

Disease resistance in animal is a polygenic trait. In an attempt to boost infection resistance, transgenic constructs containing the immunoglobulin-A (IgA) gene have been successfully introduced into pigs, sheep, and mice. The development of cattle devoid of the prion protein, which prevents infection and transmission of spongiform encephalopathies, such as scrapie and bovine spongiform encephalopathy, was a noteworthy feat. To combat mastitis, transgenic dairy cows that exude lysostaphin in their milk have been developed. Lysostaphin is an antimicrobial peptide that protects the mammary gland against infection by killing Staphylococcus aureus bacteria in a dose-dependent manner.

24.9.7.4 Improving Hair and Fiber Production

Transgenesis using the sheep wool keratin and keratin- associated protein (KAP) gene may alter the protein composition of wool fiber, resulting in fiber types with superior processing and wearing properties.

24.9.7.5 Modification of Digestion

Phytase is an enzyme that converts inorganic phosphorous to organic phosphorous, increasing the amount of phosphorous available to animals. This enzyme is generally found in ruminants; however, it is not found in monogastric animals.

The phytase enzyme is expressed in the saliva of transgenic pigs, which improves digestion and lowers feed costs in pig production.

24.9.7.6 Transgenic Animal as Bioreactor

The use of transgenic animals as bioreactors was initially focused on the mammary glands, but blood, bladder, eggs, and male accessory glands are now all considered bioreactors for medicinal proteins.

24.9.7.7 Transgenic Mammary Glands

Human milk lysozyme is a vital protein for innate immunity however commercial manufacturing of this enzyme is difficult to come by. Human lysozyme expressed in a cow’s mammary gland produces “value added” milk which is beneficial for orphan children. α1-protease inhibitor expressed in transgenic sheep (Tracy) mammary gland is purified and used for the treatment of lung emphysema. Nexia biotechnologies (Canada) has successfully transplanted the spider silk gene into goats, and the goats’ milk now contains the protein that makes up spider silk. This is known as Biosteel, the strongest fiber on the earth. This is used for manufacturing bullet-proof vests and suture silk for closing up of wounds.

24.9.7.8 Transgenic Blood

Human hemoglobin was extracted from transgenic pig blood and used to make a blood substitute for human patients.

Application in xenotransplantation: Pigs are considered as a popular source of organs for transplantation in humans since their physiology and other characteristics, such as organ size, indicate that they are among the best non-primate potential donors. The major antigen responsible for hyperacute rejection is the α₁₃-galactose (α₁,₃- Gal) epitope. The enzyme α₁₃-galactosyl transferase (α₁₃- GT) produces this epitope, although the enzyme is inactive in humans. The gene has been knocked out in pig used as organ donor by transgenesis technology.

Application as disease model: The transgenic pigs were employed as a large animal model for retinitis pigmentosa, a human eye disease. Transgenic rabbits are used as an animal model for human cardiovascular disease and atherosclerosis.

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Precise Genome Editing by Engineered Nuclease

Site-specific double-strand break is the key to precise genome editing. Several distinct classes of nucleases have been discovered and bioengineered for this purpose. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALEN), meganucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.

Learning Outcomes

• Artificial insemination: AI is the most common method of breeding intensively reared domestic livestock, such as dairy cattle, buffalo, and pigs. AI is a process by which sperm is collected from the male, processed, stored, and artificially introduced into the female reproductive tract for the purpose of conception by using means other than sexual intercourse or natural insemination.

• Sperm cryopreservation: Sperm cryopreservation helps in the propagation of animals with better genetic features and species conservation. The spermatozoa are combined with a cryoprotectant such as glycerol and a protective solution including lipoproteins, carbohydrates, and a cryoprotectant. These components aid in the preservation of membrane integrity throughout the chilling and rewarming processes.

• Sexed semen technology: Sexed semen technology is used to generate a calf of a specific sex. Sex preselection has a significant impact on the profitability of livestock-based industry. The inherent differences between features of X and Y chromosome-bearing spermatozoa are utilized for sorting of sperm.

• Spermatogonial stem cells (SSCs): SSCs comprise a subpopulation of undifferentiated spermatogonia in testis that controls the process of spermatogenesis. SSCs depend upon a specific microenvironment known as a “niche” for survival and development. SSCs are useful in animal genetics, breeding, and restoration of male fertility.

• Multiple ovulation and embryo transfer (MOET): MOET is a widely used technique for the conservation of elite genetic resources. The animal is induced for multiple ovulation through various hormonal treatment protocols and inseminated subsequently. Embryos are flushed, graded, and transferred to the recipient animal’s uterus.

(continued)

• In vitro embryo production (IVEP): In vitro embryo production (IVEP) refers to the processes of in vitro oocyte maturation (IVM), in vitro fertilization (IVF), and the early days of in vitro embryo culture (IVC). Immature oocytes are collected by ovum pick-up (OPU) or from slaughterhouse ovaries, and cultured in maturation medium. In vitro fertilization is carried out and zygotes generated are cultured for 7-8 days in vitro upto the stage of blastocyst.

• Somatic cell nuclear transfer (SCNT): is a technique by which a nucleus from a cell of the donor animal is inserted into an enucleated oocyte, and the reconstructed oocyte is allowed to grow upto embryonic development, and embryos are transferred into a recipient animal.

• Transgenesis: Transgenesis is the process of introducing foreign gene or genes of interest introduced into the animal genome. The gene (s) are transmitted and expressed by their progeny.

Exercises

Objective Questions

Q1. What are the major cryoprotectants used for the cryopreservation of sperm?

Q2. What are the sperm parameters to be considered for determining freezing rate of sperm?

Q3. Which dye is used for flow cytometry-based sex sorting of spermatozoa?

Q4. Which property of sperm cell is used for Magnetic nanoparticle (MNP)-based sperm sorting?

Q5. Which techniques are used for isolation and enrichment of spermatogonial stem cells?

Q6. How many primordial follicles are present in the ovary at the time of birth of a calf?

Q7. Which chemical is used for attachment of two demioocytes to form a couplet during hand-guided cloning?

Q8. What is coasting?

Q9. In which year cloned sheep “Dolly” was born?

Q10. What are the epigenetic modification agents used for increasing animal cloning efficiency?

Q11. Which protein is required for binding of foreign DNA with sperm in sperm-mediated transgenesis?

Q12. Give an example of non-integrating viral vector used in transgenesis.

Q13. Which antimicrobial peptide gene is transferred to mammary gland of animal for mastitis prevention?

Q14. What are the nucleases required for precise editing of genome?

Q15. Which gene is transferred for Biosteel production?

Q16. Which gene is knocked out from pig genome for using the animal as organ donor?

Q17. Which hormones are supplemented in in vitro maturation medium of oocyte?

Q18. What is the recommended post-thaw motility of cryopreserved spermatozoa for performing AI?

Q19. What is the freezing rate for slow freezing of sperm?

Q20. What are the key components of plasmid-based binary transposon system?

Subjective Questions

Q1. What are the symptoms of estrus in animals?

Q2. What are the components used as cryoprotectant? Mention their functions.

Q3. What are the major differences between X- and Y-bearing spermatozoa?

Q4. Describe the culture method of spermatogonial stem cells?

Q5. How restoration of male animal fertility can be done using spermatogonial stem cells?

Q6. Write the steps of hand-guided somatic cell nuclear transfer method.

Q7. What is “pharming”? How is it done?

Q8. How does epigenetic reprogramming help to increase the cloning efficiency?

Q9. What is the usefulness of embryo transfer in animal? Q10. How transposon-mediated transgenesis is performed? Q11. Describe the protocol of superovulation protocol in small ruminants.

Q12. Elucidate the mechanism of Cre-lox recombination system.

Q13. Describe the advanced methods of sperm sexing.

Q14. What are the applications of spermatogonial stem cells?

Q15. How transgenic technology can be applied for improving milk production and composition?

Answer to Objective Questions

A1. Glycerol, dimethylsulfoxide (DMSO), and ethylene glycol

A2. The surface-to-volume ratio of the spermatozoa and the permeability of the membrane

A3. Bis-benzimide (Hoechst 33342)

A4. Difference in zeta potential between X and Y chromosome-bearing sperm

A5. Fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS)

A6. 150,000-200,000

A7. Phytohemagglutinin

A8. The period between the last hormonal treatment and OPU

A9. 1997

A10. Trichostatin A, scriptaid, and valproic acid

A11. DNA-binding protein (DBP)

A12. Adenoviral vectors

A13. Lysotaphin

A14. Zinc finger nucleases (ZFNs), transcription-activatorlike effector nucleases (TALEN), meganucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system

A15. Spider silk gene

A16. α₁₃-Galactosyl transferase (α₁₃-GT)

A17. LH and FSH

A18. 40-50%

A19. -0.5 °C/min

A20. Transposon construct harboring the gene of interest flanked by inverted terminal repeats (ITR) and the transposase protein

Keywords for the Answer to Subjective Questions

A1. Restlessness or increased activity, vocalization, chin resting, vulva swelling, vaginal discharge, and mounting other animals

A2. Glycerol, dimethylsulfoxide (DMSO), and ethylene glycol

A3. Motility, surface charge, sperm surface antigen, DNA content

A4. Glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2)

A5. Three-dimensional microenvironment, supplementation of various growth factors, 3D-agarose gel

A6. Preparation of donor somatic cell or nuclei, recipient oocyte selection, in vitro maturation and enucleation, reconstruction of embryos, in vitro culture of embryos, and transfer

A7. The process of expressing a gene of pharmaceutical proteins in the mammary gland is termed as “pharming”

A8. DNA methylation, histone modification, genomic imprinting, and X chromosome inactivation

A9. Exploitation of female reproductive capacity (more offspring from valuable donors), import and export for valuable genetic material, development of new breeding strategies, embryo splitting, introduction of new genes into closed herds, manipulation of embryos, transgenesis

A10. Plasmid-based binary transposon system

A11. Day 1 to Day 15 intravaginal pessary (progesterone), Day 13 eCG (1200 IU), Day 17 estrus, Day 18 insemination

A12. Excision, inversion, translocation

A13. SexedULTRA™ technology of sperm sorting, Microfluidics dielectrophoretic chip-based sperm sorting, Magnetic nanoparticle (MNP)-based sperm sorting

A14. Genome editing, generating transgenic animals, restoration of fertility in male animals

A15. Creation of transgenic animal producing lactalbumin, lysozyme, lysostaphin, or other antimicrobial peptides

Animal Transgeneis

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