Animal Biotechnology FSN4409 PDF

FSN4409 FOOD BIOTECHNOLOGY ANIMAL BIOTECHNOLOGY Dr Marcus Wong 1 Overview ¡ In the past 40 years, a number of modern techniques for improving animal lines have been developed ¡ Artificial insemination (in vivo fertilization) has already had an enormous impact on t...

FSN4409 FOOD BIOTECHNOLOGY ANIMAL BIOTECHNOLOGY Dr Marcus Wong 1 Overview ¡ In the past 40 years, a number of modern techniques for improving animal lines have been developed ¡ Artificial insemination (in vivo fertilization) has already had an enormous impact on the dairy industry ¡ Other techniques such as in vitro fertilization, embryo cloning, and nuclear transplantation are becoming more prevalent ¡ Transgenic technology offers some direct medical benefits, including the availability of valuable therapeutic proteins ¡ Has the potential to provide a precise genetic route to developing disease-resistant strains of farm animals as well as strains that produce lean meat or grow more efficiently 2 Overview of GM Animals ¡ GM animals currently being developed can be placed into six different broad classes: I. investigate human diseases; II. produce industrial or consumer products; ef. worl III. produce human therapeutic proteins; of antibodys IV. enrich or enhance the animals’ interactions with humans (hypoallergenic pets); V. enhance production or food quality traits for faster growing fish, pigs that digest food more efficiently; and VI. improve animal health for disease resistance 3  11 11 11 11 Annie, Pure bred Jersey Genetically engineered calf carrying bacterial gene and cloned pigs for use in to produce lysostaphin, human organ transplants. 2000. 4  Animal Biotechnology ¡ Biotechnology in animal breeding ¡ Transgenic Animals ¡ Animal cloning ¡ Transgenic animal cell culture/ bioreactor ¡ Genome Editing https://www.precedenceresearch.com/animal-biotechnology-market 5 Biotechnology in Animal Breeding ¡ Goal of animal breeding: ¡ To generate animal products (meat, milk, eggs, wool) with enhanced quality and quantity ¡ Traditional breeding (selective breeding) ¡ Mating of selected animals following phenotypic criteria ¡ Animal breeding using biotechnology (modern) ¡ Artificial insemination ¡ In-vitro fertilization ¡ Embryo transfer ¡ Genetic maps and gene marker assist breeding 6 Artificial ↑I Insemination (AI) ¡ A process by which sperm is placed into the reproductive tract of a female for the purpose of impregnating the female by using means other than sexual intercourse ¡ The first insemination station for cattle breeding was established in Germany in 1942 ¡ AI is not costly and allows the selection of male animals with a high breeding value ¡ In most industrialized countries, more than 80% of cows become pregnant through AI 7 Advantages of Artificial Insemination ¡ Genetic Improvement: AI allows farmers to access superior genetics from bulls that may be geographically distant or prohibitively expensive to purchase. This enhances the overall genetic quality of the herd, leading to better growth rates, milk production, and disease resistance. ¡ Disease Control: By minimizing direct contact between males and females, AI reduces the risk of transmitting sexually transmitted diseases among livestock. This is particularly important in maintaining herd health. ¡ Increased Efficiency: A single bull can sire many offspring through AI, significantly reducing the number of bulls required on a farm. This not only lowers costs but also simplifies management practices. ¡ Enhanced Reproductive Management: AI allows for more precise timing of insemination, which can lead to higher conception rates when performed correctly. Proper training and techniques are essential for optimizing success rates. 8 Artificial Insemination (AI) ¡ AI involves several steps: 1. Semen collection ¡ Use of artificial vagina ¡ Are also commercially available 2. Semen storage ¡ Can be stored for indefinite periods in liquid N2 with glycerol added 3. Insemination ¡ Recto vaginal method of insemination ¡ Semen straw is loaded in a sterilized A.I. gum ¡ Inseminator will insert the gloved, lubricated left hand into the rectum, further inserted and will catch hold the cervix through rectal wall ¡ The A.I gum loaded with semen straw is passed https://www.youtube.com/watch?v=R6fYyxs_lDE 9 10 In Vitro Fertilization (IVF) ¡ Being used when artificial reproductive techniques fail ¡ blocked reproductive systems, non-responsive ovaries in the females, marginal semen quality and quantity in the male, and presence of disease ¡ The fertilization of the sperm and the egg is conducted in- vitro (outside the animal’s body) ¡ Due to advances in embryo production and cryopreservation of reproductive cells, IVF offsprings have been born in mice, rats, hamsters, cats, guinea pig, squirrels, pigs, cows, monkeys, and humans 11 Steps in In Vitro Fertilization (IVF) 1. Ovarian stimulation ¡ Hormones will be used to induce superovulation 2. Egg retrieval 3. In Vitro Fertilization 4. Selection ¡ Pre-implantation Genetic Diagnosis (PGD) procedures may be performed for embryo prior to transfer 5. Embryo transfer ¡ The embryos judged to be the "best" are transferred to the uterus 12 In vitro fertilization 13  14 Fertilization Process Pronucleus Embryo Transfer (ET) ¡ Embryo transfer (ET) to a surrogate mother allows livestock progenies to be produced from a superior female ¡ Selected females are induced to superovulate hormonally and inseminated ¡ Week-old embryos are flushed out of the donor’s uterus, isolated, examined microscopically for number and quality, and inserted into the lining of the uterus of surrogate mothers ¡ Increases reproductive rate of selected females ¡ Reduces disease transfer and facilitates the development of rare and economically important genetic stocks 15  Embryo Transfer https://www.repro360.com.au/reproductivetechnologies/et 16 Transgenic Animals ¡ Definition: Animals that have one or more genes from a different sources such as human or microorganisms, or genes that have been altered or specially assembled, inserted into their genome ¡ The most famous transgenic animal is the “supermouse”, created in the early 1980s by inserting a human growth hormone gene in mouse genome. The offspring were twice as large as their non-transgenic littermates ¡ Fostered expectations that farm animal would soon be developed that had boosted levels of growth hormone 17 Transgenic Animals ¡ Numerous attempts in creating transgenic animals ¡ Such animal might have leaner meat, better feed efficiency and faster growth ¡ Although transgenic pigs have these improvements, but they are also susceptible to a number of abnormalities, particularly in skeletal development ¡ Likely related to the increased expression of growth hormone, and not to other factors ¡ Still under development 18 The Infamous "Beltsville Pig" ¡ In 1989, researchers added a gene into the DNA of a pig that would produce a human growth hormone ¡ The expectation was that the animal would grow faster and be leaner than normal pigs ¡ The researchers were successful: weight gain increased by 15%, feed efficiency by 18%, and carcass fat was reduced by 80% ¡ But the animals suffered from several unanticipated health problems ¡ Kidney and liver problems, uncoordinated walking, bulging eyes, gastric ulcers, heart disease and pneumonia 19 Basic Procedure for Production of Transgenic Animals 1. Identification of target gene 2. Cloning of gene 3. Production of a suitable gene construct 4. Transfer of gene ¡ Most important and bottle neck step ¡ Microinjection method – first method ¡ Embryonic Stem Cells 5. Proof of integration of the foreign gene 6. Proof of expression (mRNA, protein) 7. Demonstration of transmission (inheritance) 8. Selective breeding 20 Methodology ¡ Step 1 – Construction of a transgene ¡ The transgene should consist of: ¡ Promoter, gene to be expressed and termination sequence ATG TAA Promoter Gene Terminator > - diff animals he diff , promoter ¡ Promoter of gene > - control expression ¡ a region of DNA where transcription of a gene initiates ¡ adjacent and typically upstream (5’) of the sense strand of the regulated gene ¡ Control the attachment of RNA polymerase to DNA and are directly responsible for the amount of transcript generated ¡ Terminator ¡ A section of nucleic acid sequence that marks the end of gene during transcription 21 Methodology Step 2 – Introduction of foreign gene into the animal > - fertilized eff. ¡ Microinjection method > - blastocyst ¡ Embryonic stem cell method ¡ Retro Virus 22 Microinjection Method t usually ¡ A female animal is superovulated and eggs are collected ¡ The eggs are fertilized in-vitro ¡ The transgene in a solution is injected into the male pronucleus using a micropipette ¡ Eggs with the transgenes are kept overnight in an incubator to develop to a 2-cell stage At ¡ The eggs are then implanted into the uterus of a pseudo- pregnant female (female which has been mated with a vasectomized male the previous night) 23 Microinjection Method ¡ Single fertilized egg ¡ Microinjection of new DNA into pronucleus ¡ Implanted into foster mother ¡ Further breeding for homozygous expression 24  Stem Cells ¡ Able to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types ¡ Embryonic stem cells isolated from the inner cell mass of blastocysts which differentiate into all of the specialized embryonic tissues ¡ Adult stem cells found in adult tissues which act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues 25 Embryonic Stem Cell Method ¡ Transgenic animals can be created by manipulating embryonic stem (ES) cells ¡ Transgene is incorporated into the ES cell by § Microinjection § By a retro virus § By electroporation § Transgenic stem cells are grown in vitro § Then they are inserted into a blastocyst and implanted into a host’s uterus to grow normally § The animal initially produced from is usually a genetic chimera of chromosome expressed and all >2 - diff types are 26 Embryonic Stem Cells ¡ ES are cultured and subjected to DNA- mediated gene transfer (DMGT) or other manipulations ¡ Cells with the desired genetic alteration are then inserted into blastocyst cavities, whereupon they resume normal development and produce a genetically mosaic mice ¡ Founders, are then bred to pass on the gene, provided the ES cells differentiate into sperm. The F1 hybrid then carries the new gene in all cells (bottom) > - selective breeding Chimera > - homogenous ILAR Journal, Volume 38, Issue 1, 1997, Pages 32–41, 27 Retro Virus no control on where the insertion is ¡ Retroviruses can infect early embryos and insert proviral DNA copies of their RNA genomes into the mouse chromosome ¡ Expose embryos to medium containing recombinant retroviruses ¡ Integration of a single copy of the donor gene ¡ Gene expression in such viral constructs is often inefficient 28 29 Methodology ¡ Step 3: Screening for transgenic positives ¡ Transgenic progenies are screened by PCR to examine the site of incorporation of the gene ¡ Some transgenes may not be expressed if integrated into a transcriptionally inactive site. ¡ Step 4: Further animal breeding is done to obtain maximal expression. ¡ Heterozygous offsprings are mated to form homozygous strains. 30 https://youtu.be/V70Uoi672-c?t=52 31 Problems ¡ Multiple insertion which leads too much proteins production ¡ More copies, more transcripts, more proteins ¡ Insertion into an essential gene results in lethality supress the gene - X expression ¡ Insertion into a gene leading to gene silencing ¡ Insertion into a different area can affect the gene regulation 32 Transgenic Animals in Agriculture ¡ Agriculture exploitation of transgenic animal technology lags behind applications in biomedicine 1. Carcass composition ¡ Improvement in growth rate, feed conversion and body composition ¡ Transgenic pig carrying desaturase gene, which will increase the non-saturation fatty acid ¡ Commercialization postponed due to lack of public acceptance 2. Lactation ¡ Increase caseins, express human proteins in cow milk ¡ Reduce lactose and butterfat ¡ Proposed stage 33 Transgenic animals in Agriculture 3. Wool production ¡ Increase cysteine synthesis ¡ Wool keratin protein ¡ Evaluation stage 4. Environmentally friendly farm animals ¡ Transgenic pigs carrying phytase gene ¡ Reduce phosphorus output by 75% 5. Disease-resistant transgenic animals ¡ Influenza resistant - Mx protein ¡ Overall resistant to infection – IgA ¡ Visna virus (causes encephalitis and chronic pneumonitis in sheep) resistant sheep – visna virus envelope proteins 34 Real-Life Example: Transgenic Fish ¡ Transgenic fish of various species of salmon, tilapia, channel catfish and others actively investigated ¡ No transgenic fish have been approved for producing food in the US and world until 2015 ¡ Limitation of current technology for transgenic fish ¡ Transgene limits to short gene long will interfere gene expression ¡ Stability of genetic modification in breeding population ¡ Control of expression of modified genes (Promoters) ¡ Antimicrobial resistant genes ¡ Biocontaminant and environmental concern 35 Real-Life Example: Transgenic fish ¡ Salmon are difficult to rear in aquaculture because of the two distinct phases of their life cycle ¡ Juvenile rearing ¡ Require cold freshwater (which leads to slow growth rates) or heated freshwater (which is expensive) ¡ Then transferred to saltwater ¡ Naturally salmon are slow growers, in the first year of life they may achieve a weight of between 20-30 grams ¡ Increased levels of growth hormone shorten the juvenile rearing period, thus cutting costs of production 36 This undated photo provided by AquaBounty Technologies shows two same-age salmon, a genetically modified salmon, rear, and a non- genetically modified salmon, foreground. 37 How does AquAdvantage Salmon work? ¡ The growth hormone from Chinook salmon is inserted into fertilised Atlantic salmon eggs ¡ Allows the Atlantic salmon to grow more like a trout, reaching desired market weight in a much faster time ¡ The Chinook salmon's growth promoting gene allows it to grow in less hospitable environments Atlantic salmon Sea trout 38 Creation of AquAdvantage ¡ Recombinant DNA inserts from three sources were used for the final construct (opAFP-GHc2): 1. Ocean Pout Anti-Freeze Protein (opAFP) Regulatory (non- coding) Sequences ¡ The upstream (5’) and downstream (3’) regulatory sequences used in the opAFP-GHc2 construct were obtained from a genomic isolate of a Type III anti-freeze protein (AFP) gene from the ocean pout (op) 2. Chinook Salmon GH Coding Sequence ¡ The Chinook salmon GH gene was identified and isolated from a complementary DNA (cDNA) library prepared using pituitary gland of Chinook salmon. This cDNA is full-length and encodes a single, mature hormone. 3. Synthetic linkers ¡ Two synthetic DNAs corresponding to the 5’ untranslated regions (UTRs) were prepared using established sequences of the Chinook salmon GH-1 and the ocean pout AFP 39 Benefits of AquAdvantage ¡ Faster growth - market weight of around 2-3kgs can be achieved in 18 to 24 months compared to 3 years ¡ AquAdvantage salmon can be grown domestically. This provides a number of benefits: > - avoid escape to the wild 1. Producing fish in closed systems would dramatically reduce transport costs and improve the whole supply chain 2. Producing more salmon in containment will reduce pressure on wild fish stocks 3. Allowing production of salmon close to populations will mean that a fresh supply of fish is available at all times 40 Concerns on AquAdvantage ¡ Environmental issues and Consumer health issues ¡ AquAdvantage salmon may escape from captivity and may result in: ¡ Breeding with wild salmon ¡ Outcompete wild salmon for natural resources Genetically modified salmon are in tanks at the AquaBounty farm in Waltham, Massachusetts 41 Concerns on AquAdvantage ¡ To relieve those concerns: ¡ All AquAdvantage salmon are sterile females, there is no chance of them breeding with wild salmon ¡ A number of other independent studies have confirmed that AquAdvantage salmon are no threat to the environment ¡ AquAdvantage salmon are domesticated and may not be able to compete in the wild ¡ Preliminary results also say that AquAdvantage salmon struggle when feed is limited, making them less competitive than their close relations, the Atlantic salmon 42 43 Example - Enviropig ¡ Phosphorous from animal manure is a nutrient for plants that becomes a pollutant if there is too much of it for crops to absorb, and the excess runs off into streams and lakes ¡ Enviropig™ - genetically engineered pig to excrete less phosphorous in its feces ¡ Developed by researchers at the University of Guelph in Ontario in 2001 ¡ The goal of the GM Enviropig™ is to provide intensive livestock operations (factory farms) with a product to reduce the amount of polluting phosphorous they produce 44 Example - Enviropig ¡ Genetically engineered to produce the enzyme phytase in its salivary glands ¡ Enable more effective digestion of phytate, the phosphorus found in pig feed ¡ Scientists inserted a transgene sequence that includes an E. coli bacteria phytase gene and a mouse promoter gene sequence ¡ Enviropig™ could contain 30 to 70.7% less phosphorus ¡ Approval Status: ¡ Reproduction and Exportation granted ¡ For use as food still pending for approval in Canada and other countries ¡ Research stopped due to public’s objection 45 Animal Cloning ¡ Cloning is an assisted reproductive technology which allows the production of offspring that is genetically identical to the single donor animal ¡ Animals being cloned offer superior production traits such as: ¡ Consistent and reliable genotype ¡ High quality or quantity of meat or milk ¡ Superior body conformation ¡ Reproductive soundness ¡ Inherent resistance to disease 46 China’s first ‘copy cat’ 47 Somatic Cell Nuclear Transfer ¡ The technique used in animal cloning ¡ Defined as the fusion of the nuclei (or entirely) of diploid donor cells with unfertilized, enucleated oocytes. -animal wants to clone somesticle 48 49 Animal Cloning ¡ In higher animals, genetically identical clones are rare ¡ Homozygous twins in humans is only 0.3% ¡ An egg is taken from the female donor, and its nucleus is removed with micropipette ¡ The Go phase of the cell cycle of a somatic cells is induced and the cell is fused to the enucleated egg cell ¡ The resulting diploid cells are developed to be the embryonic stage either in cell culture or in the oviduct of a sterile female, and will be transferred into a foster mother 50 Difficulties in Cloning Process ¡ Certain gene regions have been blocked in fully developed somatic cell ¡ Donor cells have suffered some previous damage ¡ The interaction between denucleated cell and injected nucleus might have problem ¡ 277 enucleated eggs and a total of 27 embryos that were derived from them were used in creating Dolly ¡ Environmental factors also contribute to the differences in phenotypes development ¡ Position in the embryo, food intake of the surrogate mother ¡ Dolly is the product of the nuclear DNA of the donor plus mitochondrial DNA from the egg cell 51 Biomedical Application of Transgenic Animals ¡ Animal bioreactor for pharmaceutical products ¡ Production of human recombinant proteins from mammary gland of transgenic animals ¡ Antithrombin III (ATIII) ¡ Antibody production on transgenic animals ¡ Monoclonal antibodies from mammary gland and blood of transgenic animals ¡ Blood replacement ¡ Functional human hemoglobin from transgenic swine ¡ Xenotransplantation of porcine organs to human patients ¡ Farm animals as models for human diseases 52 Animal Cell Culture ¡ Definition: Massive growth of animal cells in vitro and use these cells to produce food and biological substances ¡ Rationale: used to produce some special functional proteins that can’t be produce by microorganisms ¡ Technology: Easier and simpler than transgenic animals 53 Transgenic Animal Cell Culture Application ¡ Food industry ¡ Potential for food production ¡ Production of functional food ¡ Medical field ¡ Vaccine – animal food and mouse diseases, herpes virus, Hepatitis B ¡ α, β, γ,- interferon ¡ Monoclonal antibody ¡ Stem cell research ¡ Other recombinant proteins 54 Lab Grown Meat BBC, 2-Dec-2020 NY Times, 5-Aug 2013 Bloomberg, 10-Dec-2020 55 Lab Grown Meat 56 57 Genome Editing ¡ Genome editing is a group of technologies that give scientists the ability to change an organism’s DNA ¡ Allows genetic material to be added, removed, or altered at particular locations in the genome ¡ Usually achieved using engineered nucleases also known as molecular scissors ¡ Different from genetic modification ¡ Because gene editing is more precise, those working in the area believe that it is much less likely to lead to unanticipated side effects 58 CRISPR cno need umb each step ¡ Short for: Clustered Regularly Interspaced Short Palindromic Repeats ¡ CRISPR-Cas9 Kenzyme was adapted from a naturally occurring genome editing system in bacteria ¡ The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays ¡ Allow the bacteria to "remember" the viruses ¡ If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA ¡ The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus 59 CRISPR 60 CRISPR – Working Principle name cas9 cutting Palindromic Sequence: Sequence in one strand is the same as the complementary sequence of the other strand when read from the same direction on both the strands, either 5’ to 3’ or 3’ to 5’ Important in formation of gRNA 61 CRISPR-Cas9 Introduces a break at target 62  Application of CRISPR….and more to come Cell. 2014 Jun 5;157(6):1262-78. doi: 10.1016/j.cell.2014.05.010. 63 Genome Editing in Disease Resistant Animals ¡ Many animal viruses are difficult to control using conventional methods (e.g. vaccination) ¡ Transgenic technology could change the animal’s genome so that it can no longer be infected ¡ May be an effective approach to control viruses (e.g. the virus that causes foot-and-mouth disease) One of the first signs of FMD is excessive salivation and lesions on the tongue and hooves 64 Genome Editing in Disease Resistant Animals ¡ For example, viruses get into host cells through binding to specific receptors ¡ Alteration of removal of such receptors thru transgenic tech might render the animals disease resistant ¡ Though it might adversely affect the animal’s metabolism or development because cell surface proteins always have a function of some sort ¡ Any successful cases? 65 Genome Editing - PRRS Resistant Pigs s 111 Is1 Ill 11 11 11/ 1 111 Ill 66 Example: PRRS Resistant Pigs ¡ Porcine Reproductive and Respiratory Syndrome ¡ PRRSV, a (+) ssRNA Virus ¡ Infected pigs may present with symptoms involving inappetence, fever, lethargy, and respiratory distress ¡ Cause a partial displacement of the placenta in pregnant sow, leading to full abortions or to death and mummification of fetuses in utero ¡ Cause diarrhea and severe respiratory distress caused by lesions in the lung in young piglets ¡ >$650m are lost annually to pork producers in the United States alone 67 Example: PRRS Resistant Pigs > - X infection > X - bind > - encode receptor binds wI virus ¡ Researchers used gene editing to delete a small region of pig DNA ¡ Prevents the PRRS virus from attaching on pig cells ¡ The scientists then exposed four of their gene-edited pigs to the PRRS virus and none became ill ¡ No foreign genes were inserted into the pig but just a tiny section of the DNA is snipped out ¡ The animals are not weakened or affected in any other way by the process ¡ The genetic edit is permanent, so disease resistance will be passed down the generations through natural breeding 68  Example: PRRS Resistant Pigs ¡ Researchers adopted CRISPR-Cas9 system to perform the precise genome editing CD163 is the receptor for PPRSV Deletion of one exon results in complete K/O > - A translated protein abnormal > - protein structure - guide RNA Guide RNA and Cas9 mRNA were introduced into zygote by Microinjection PLoS Pathog (2017).13(2): e1006206. 69  Other Applications of CRISPR in Food https://www.greenqueen.com.hk/scifi-foods-crispr- cultivated-beef/ https://news.wsu.edu/press-release/2023/05/01/wsu-first- university-to-put-gene-edited-livestock-into-human-food- supply/?utm_medium=email&utm_source=rasa_io&utm_camp aign=newsletter 70 https://www.nature.com/articles/d41587-021-00026-2 Transgenic Animals: Potential Benefits and Concerns ¡ On one side: ¡ Specificity - Accurate choose wanted traits and minimize unwanted ¡ Speed of breeding - Desire traits established in one generation ¡ Flexibility - New (cross species) traits possible ¡ Economy - Feed efficiency and fewer treatment ¡ On the other side: ¡ Animal welfare - Transgenes upset genome expression inverted disease e ¡ Virus transfer - Particular concern for animal as tissue donor. ¡ Dissemination - Transgenes release to wild population ¡ Food safety concern 71 Summary ¡ Technologies to generate transgenic animals and control transgene expression have made significant progress ¡ More than 90% transgenic animals are used to study gene function and mechanisms of action ¡ Many are for studying human diseases ¡ Transgenic animals in biomedical application made some breakthrough ¡ Development in transgenic farm animals has limited due to the public resistance to GM food ¡ Transgenic fish is the first transgenic food animals, if not the last 72