Pharmaceutical and Biotechnological Applications of Transgenic Technology

A transgenic animal is a genetically modified species in which researchers have modified an existing gene or genes by genetic engineering techniques. Genetic modification involves the mutation, insertion, or deletion of genes. Mouse is the most widely used mammalian species for creating transgenic lines. There are two types of transgenic animals: (i) gene deleted (“knock-out”) and (ii) gene overexpressed (“knock-in”). The loss or gain of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable characteristics. Knockout mice are key animal models for studying the role of genes which have been sequenced but whose functions have not been determined. They include constitutive knockouts (gene deleted since birth) and conditional knockout (gene turned off later after birth). The first knockout mouse was created in 1989 by Mario Capecchi, Martin Evans, and Oliver Smithies, for which they were awarded the 2007 Nobel Prize in Physiology or Medicine. Transgenic mouse models have revolutionized the biomedical research and provided a power tool for understanding health and disease. Transgenic animals have been created for bulk production of biotechnology and pharmaceutical products. In 2009, the FDA approved the first human biological drug ATryn, an anticoagulant extracted from the transgenic goat's milk. The recently discovered CRISPER gene editing technology is providing new frontiers in correcting abnormal genes and hopefully provide cures for genetic diseases in the future.

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Expression Systems and Species Used for Transgenic Animal Bioreactors

BioMed Research International ◽

10.1155/2013/580463 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 18

Author(s):

Yanli Wang ◽

Sihai Zhao ◽

Liang Bai ◽

Jianglin Fan ◽

Enqi Liu

Keyword(s):

Recombinant Proteins ◽

Mammalian Cells ◽

Mammalian Species ◽

Therapeutic Proteins ◽

Transgenic Animals ◽

Mammary Glands ◽

Transgenic Animal ◽

High Fertility ◽

Expression Systems ◽

Specific Expression

Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

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Genetics and Public Health

Examining the Causal Relationship Between Genes, Epigenetics, and Human Health - Advances in Bioinformatics and Biomedical Engineering ◽

10.4018/978-1-5225-8066-9.ch019 ◽

2019 ◽

pp. 529-546

Keyword(s):

Public Health ◽

Health Service ◽

Genetic Diseases ◽

Health Interventions ◽

Biological Knowledge ◽

Cellular Mechanisms ◽

Health Concerns ◽

Environmental Models ◽

The Social ◽

Health And Disease

This chapter wraps up by discussing the crucial role played by public health specialists who must reconcile traditional public health concerns of health inequality and equity with safe and effective health interventions and diagnostics that meet individual health needs. Since most genetic diseases in the realm of public health are an interplay of different genetic, lifestyle, and environmental factors, genomic science has given greater emphasis to the importance of molecular and cellular mechanisms in health and disease. New biological knowledge must be integrated with the social and environmental models to improve health at individual and population levels. Public health specialists must now be able to integrate genome-based knowledge into public health in a responsible, ethical, and effective way and anticipate the increase in the health service requirements likely to occur in the future. The foundational pillars of bioethics (beneficence, non-maleficence, autonomy, and justice) must be protected by all public health stakeholders.

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Abstract 14: Central Nervous System Deletion of the Bbs1 Gene Causes Obesity and Hypertension in Mice

Hypertension ◽

10.1161/hyp.62.suppl_1.a14 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Deng-Fu Guo ◽

Donald A Morgan ◽

Charles C Searby ◽

Darryl Y Nishmura ◽

Val C Sheffield ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Body Weight ◽

Arterial Pressure ◽

Energy Homeostasis ◽

Autosomal Recessive Disorder ◽

Conditional Knockout ◽

Peripheral Tissues ◽

Knock Out ◽

Obesity Phenotype

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder with several features including obesity and hypertension. Systemic knock-out mouse models lacking expression of Bbs2, Bbs4 and Bbs6 genes, and Bbs1 M390R knock-in recapitulated many of the BBS phenotypes including obesity. However, the role and contribution of different tissues to the various phenotypes associated with BBS including obesity and hypertension remains unclear. To address this, we generated a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed. Cre-mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in the central nervous system (CNS) affects body weight and arterial pressure. Breeding Bbs1 flox with nestin Cre mice created mice deficient in Bbs1 gene only in the CNS as indicated by the loss of Bbs1 gene expression (by RT-PCR) in the hypothalamus, hippocampus, cortex and brainstem, but not in peripheral tissues such as adipose tissue, liver, kidney and skeletal muscle. Importantly, Bbs1 flox /nestin Cre mice display an obesity phenotype as indicated by the increased (P<0.05) body weight (40±1 g vs. 31±1 g in controls) and fat mass measured by MRI (23±2 g vs. 9±1 g in controls) in 25 weeks old mice. We found that the obesity phenotype in Bbs1 flox /nestin Cre mice is due to both an increase (P<0.05) in food intake (4.0±0.2 g vs. 3.1±0.3 g in controls) and reduction in energy expenditure as indicated by the decreased (P<0.05) O 2 consumption (2.8±0.3 mL/100g/min vs. 3.2±0.2 mL/100g/min in controls) and heat production (8.3±0.8 kcal/kg/h vs. 9.4±0.7 kcal/kg/h in controls). These results indicate that hyperphagia and low metabolic rate explain the development of obesity in Bbs1 flox /nestin Cre mice. Finally, we assessed by radiotelemetry the consequence on arterial pressure of ablating the Bbs1 gene throughout the CNS. Interestingly, CNS deletion of the Bbs1 gene recapitulates the hypertension phenotype of BBS as indicated by the elevated mean arterial pressure in Bbs1 flox /nestin Cre (123±3 mmHg) relative to littermate controls (112±4 mmHg, P=0.02). These findings demonstrate that Bbs genes in the CNS are critical for energy homeostasis and arterial pressure regulation.

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Genetics and Public Health

Research Anthology on Public Health Services, Policies, and Education ◽

10.4018/978-1-7998-8960-1.ch029 ◽

2021 ◽

pp. 630-646

Author(s):

Oscar J. Wambuguh

Keyword(s):

Public Health ◽

Health Service ◽

Genetic Diseases ◽

Health Interventions ◽

Biological Knowledge ◽

Cellular Mechanisms ◽

Health Concerns ◽

Environmental Models ◽

The Social ◽

Health And Disease

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Inactivation of porcine interleukin-1β results in failure of rapid conceptus elongation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718004115 ◽

2017 ◽

Vol 115 (2) ◽

pp. 307-312 ◽

Cited By ~ 12

Author(s):

Jeffrey J. Whyte ◽

Ashley E. Meyer ◽

Lee D. Spate ◽

Joshua A. Benne ◽

Raissa Cecil ◽

...

Keyword(s):

Gene Editing ◽

Mammalian Species ◽

Interleukin 1Β ◽

Corpora Lutea ◽

Morphological Transition ◽

Wild Type ◽

Morphological Transformation ◽

Proinflammatory Response ◽

Knock Out ◽

Estrogen Synthesis

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1β, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2−/−) conceptuses. Although the morphological transition of IL1B2−/− conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

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Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures

Reproduction Fertility and Development ◽

10.1071/rd04117 ◽

2005 ◽

Vol 17 (2) ◽

pp. 113 ◽

Cited By ~ 23

Author(s):

Poul Maddox-Hyttel ◽

Jakob O. Gjørret

Keyword(s):

Stem Cells ◽

Adult Stem Cells ◽

Germ Line ◽

Current Knowledge ◽

Mammalian Species ◽

Embryonic Stem ◽

Transgenic Animals ◽

Great Expectations ◽

Safety Issues ◽

Germ Line Transmission

Current knowledge on the biology of mammalian embryonic stem cells (ESC) is stunningly sparse in light of their potential value in studies of development, functional genomics, generation of transgenic animals and human medicine. Despite many efforts to derive ESC from other mammalian species, ESC that retain their capacity for germ line transmission have only been verified in the mouse. However, the criterion of germ line transmission may not need to be fulfilled for exploitation of other abilities of these cells. Promising results with human ESC-like cells and adult stem cells have nourished great expectations for their potential use in regenerative medicine. However, such an application is far from reality and substantial research is required to elucidate aspects of the basic biology of pluripotent cells, as well as safety issues associated with the use of such cells in therapy. In this context, methods for the derivation, propagation and differentiation of ESC-like cultures from domestic animals would be highly desirable as biologically relevant models. Here, we review previously published efforts to establish bovine ESC-like cells and describe a procedure used in attempts to derive similar cells from bovine Day 12 embryos.

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Bupropion and Citalopram in the APP23 Mouse Model of Alzheimer's Disease: A Study in a Dry-Land Maze

International Journal of Alzheimer s Disease ◽

10.1155/2012/673584 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Katharina L. Neumeister ◽

Matthias W. Riepe

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Spatial Learning ◽

Transgenic Animals ◽

Transgenic Animal ◽

Wild Type ◽

Dry Land ◽

Psychomotor Activity ◽

Model Of Alzheimer’S Disease ◽

Resting Time

Background. Incipient Alzheimer's disease is often disguised as depressive disorder. Over the course of AD, depressive symptoms are even more frequent. Hence, treatment with antidepressants is common in AD. It was the goal of the present study to assess whether two common antidepressants with different mechanisms of action affect spatial learning in a transgenic animal model of Alzheimer's disease.Methods. We assessed spatial memory of male wild-type and B6C3-Tg(APPswe,PSEN1dE9)85Dbo (APP23) transgenic animals in a complex dry-land maze. Animals were treated with citalopram (10 mg/kg) and bupropion (20 mg/kg).Results. Moving and resting time until finding the goal zone decreased in 4.5-month-old sham-treated wild-type animals and, to a lesser extent, in APP23 animals. Compared with sham-treated APP23 animals, treatment with bupropion reduced resting time and increased speed. On treatment with citalopram, moving and resting time were unchanged but speed decreased. Length of the path to the goal zone did not change on either bupropion or citalopram.Conclusion. Bupropion increases psychomotor activity in APP23 transgenic animals, while citalopram slightly reduces psychomotor activity. Spatial learning per se is unaffected by treatment with either bupropion or citalopram.

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Use of score sheets for welfare assessment of transgenic mice

Laboratory Animals ◽

10.1258/0023677011911859 ◽

2001 ◽

Vol 35 (4) ◽

pp. 379-389 ◽

Cited By ~ 30

Author(s):

M. van der Meer ◽

A. Rolls ◽

V. Baumans ◽

B. Olivier ◽

L. F. M. van Zutphen

Keyword(s):

Early Detection ◽

Transgenic Mice ◽

Large Scale ◽

Transgenic Animal ◽

Daily Routine ◽

Careful Monitoring ◽

Welfare Assessment ◽

Daily Care ◽

Routine Monitoring ◽

Transgenic Lines

The use of transgenic mice has increased dramatically in recent years and continues to increase further. However, because transgenesis may alter a balanced genotype and produce unpredictable effects, careful monitoring of health and welfare of the transgenic animal is advised. The present study assessed the feasibility of the use of score sheets for monitoring transgenic mice, as part of daily routine, in a transgenic unit. The score sheets used were based on parameters which are sensitive and easy to determine. The score sheets were used by two animal technicians and a thorough evaluation showed that the score sheets, as described in this paper, are useful for routine monitoring in a transgenic unit and may result in the early detection of animal welfare problems. However, notwithstanding the limited number of parameters included and the restricted age-span covered by the screening, the monitoring system was considered to be time consuming. Large-scale implementation of such a scoring system during the first weeks of life would increase daily care time by at least 15-20 min for an average litter of 4-6 pups. Nevertheless, the use of score sheets seems to be a prerequisite for monitoring the animal's welfare in the course of producing transgenic lines.

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319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab319 ◽

2015 ◽

Vol 27 (1) ◽

pp. 248

Author(s):

N. G. Canel ◽

R. J. Bevacqua ◽

M. I. Hiriart ◽

N. Chavez Rabelo ◽

L. S. Almeida Camargo ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Transgenic Animals ◽

Transgenic Animal ◽

Egfp Expression ◽

Control Group ◽

Randomisation Test ◽

Blastocyst Quality ◽

Fragmented Nucleus ◽

Vitro Development

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals. Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both

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Cosmc is required for T cell persistence in the periphery

Glycobiology ◽

10.1093/glycob/cwz054 ◽

2019 ◽

Vol 29 (11) ◽

pp. 776-788 ◽

Cited By ~ 1

Author(s):

Christopher E Cutler ◽

Mark B Jones ◽

Alicia A Cutler ◽

Amanda Mener ◽

Connie M Arthur ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Conditional Knockout ◽

Cell Maturation ◽

Direct Role ◽

Knock Out ◽

Cell Counts ◽

Antigen Loss ◽

Spleen And Lymph Nodes ◽

T Cell Maturation

Abstract T lymphocytes, a key arm of adaptive immunity, are known to dynamically regulate O-glycosylation during T cell maturation and when responding to stimuli; however, the direct role of O-glycans in T cell maturation remains largely unknown. Using a conditional knockout of the gene (C1GalT1C1 or Cosmc) encoding the specific chaperone Cosmc, we generated mice whose T cells lack extended O-glycans (T cell conditional Cosmc knock out or TCKO mice) and homogeneously express the truncated Tn antigen. Loss of Cosmc is highly deleterious to T cell persistence, with near-complete elimination of Cosmc-null T cells from spleen and lymph nodes. Total T cell counts are 20% of wild type (WT), among which only 5% express the truncated glycans, with the remaining 95% consisting of escapers from Cre-mediated recombination. TCKO thymocytes were able to complete thymic maturation but failed to populate the secondary lymphoid organs both natively and upon adoptive transfer to WT recipients. Our results demonstrate that extended O-glycosylation is required for the establishment and maintenance of the peripheral T cell population.

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