Transgenic animals have foreign DNA (transgene) stably integrated into their genome in all cells. Production: microinjection of DNA into fertilised egg pronucleus, OR retroviral vector, OR embryonic stem cell technology, OR CRISPR/Cas9. Applications: Molecular pharming (biopharmaceutical production in milk/blood/eggs). Disease models (mice with human disease genes). Xenotransplantation (pig organs humanised for human transplantation). Nutrition improvement (Golden rice, omega-3 pigs). Safety testing of vaccines, toxicology. Key examples: AAT (alpha-1-antitrypsin) in transgenic sheep milk → emphysema treatment. Antithrombin III (Atryn) in transgenic goat milk → hereditary antithrombin deficiency treatment. C1 inhibitor in transgenic rabbit milk → hereditary angioedema. Recombinant insulin from bacteria (Humulin) — not transgenic animal.

AAT (alpha-1-antitrypsin, also called alpha-1-proteinase inhibitor) is produced mainly by hepatocytes (liver cells). It is the most abundant serine protease inhibitor in human plasma. Target enzyme: neutrophil elastase. Elastase: a powerful enzyme released by neutrophils (white blood cells) during inflammation to degrade bacterial proteins and destroy pathogens. Without AAT: elastase also destroys elastin and other proteins in the lung parenchyma. AAT inhibits elastase → protects alveolar walls from proteolytic destruction. This protease-antiprotease balance is critical in the lungs. Smoking disrupts this balance: cigarette smoke oxidises the active site methionine of AAT → inactivates AAT → neutrophil elastase uncontrolled → alveolar destruction → emphysema. Even in non-deficient individuals, smoking destroys AAT function.

Emphysema is defined as abnormal permanent enlargement of air spaces distal to the terminal bronchioles, with destruction of alveolar walls, without obvious fibrosis. Pathogenesis: Inflammation (from smoking, infection) → neutrophils and macrophages recruited to lung → release elastase and other proteases → destroy elastin and collagen in alveolar walls → alveolar walls break down → abnormally large air spaces. Loss of elastic recoil: alveoli destroyed → lungs lose their elastic tissue → lungs become hyperinflated (cannot fully deflate). Types: Centriacinar emphysema: affects respiratory bronchioles, predominantly in upper lobes. Associated with smoking. Panacinar emphysema: affects entire acinus (including alveoli) diffusely. Associated with AAT deficiency. Clinical features: progressive breathlessness (dyspnoea), barrel chest, decreased breath sounds, pursed-lip breathing (to generate positive end-expiratory pressure and prevent airway collapse). Diagnosis: chest X-ray (hyperinflation, flat diaphragm), CT scan (best), spirometry (obstructive pattern).

AAT deficiency is the most common serious hereditary liver/lung disease in adults in Western populations. Genetics: SERPINA1 gene on chromosome 14. Normally: M allele (wild type) → normal AAT. Common pathological alleles: Z allele (Glu342Lys mutation): most severe. ZZ genotype → AAT polymerises in ER of hepatocytes → cannot be secreted → accumulates in liver → liver damage + very low serum AAT levels. SS allele: milder reduction. MM: normal. PiZZ (homozygous Z): serum AAT <15% normal → severe emphysema (onset 30s-40s if non-smoking, earlier if smoking) + liver cirrhosis. Prevalence: approximately 1 in 3000-5000 in Northern Europeans. Treatment: weekly IV infusions of pooled human AAT (augmentation therapy). Transgenic production (pharming) provides an alternative source.

Molecular pharming (biopharming): using transgenic animals to produce therapeutic proteins in easily harvested biological fluids (milk, blood, urine, eggs). Why milk is ideal: mammary glands are natural protein factories — cow produces ~10 litres/day, sheep ~1-2 litres. Large amounts of protein can be produced. Proteins are correctly folded and post-translationally modified. Easy to harvest by milking. Protein can be purified from milk. Method: human therapeutic gene + mammary-specific promoter (from milk protein gene: beta-casein, whey acidic protein) → injected into fertilised egg → transgenic founder animal → bred to establish herd. Approved pharming products: Atryn (antithrombin III from goat milk, GTC Biotherapeutics, 2006 — first approved). Ruconest (C1 esterase inhibitor from rabbit milk, PharmaBio/Pharming Group). AAT from sheep milk — clinical trials. Advantages: lower cost than cell culture. Correct glycosylation. Scalable. Disadvantages: long development time (need to breed transgenic herd), animal welfare concerns, prion/virus contamination risk.

Transgenic and knockout mice have revolutionised biomedical research as disease models. Knockout mice: specific gene deleted → study gene function. Transgenic mice: human disease gene inserted → mouse develops human disease → study disease + test drugs. Key models: Oncomouse (Harvard mouse): first patented animal. Contains activated human ras oncogene → develops tumours → cancer research model. SCID mice (severe combined immunodeficiency): lack functional T and B cells → accept human tumour grafts → xenograft cancer models. Alzheimer mouse models: express mutant human APP (amyloid precursor protein) and presenilin → develop amyloid plaques. ApoE knockout mice: develop atherosclerosis. Cystic fibrosis mice: CFTR gene deletion. Huntington disease mice. These models allow: understanding disease mechanisms, identifying therapeutic targets, pre-clinical drug testing before human trials.

Recombinant DNA technology produces many therapeutic proteins: Insulin (Humulin): first recombinant therapeutic protein. Human insulin gene in E. coli/S. cerevisiae. Approved 1982. Human growth hormone (HGH, Somatropin): from E. coli. Treats growth deficiency. Previously from cadaver pituitaries (CJD risk). Factor VIII (recombinant): haemophilia A treatment. Produced in CHO cells. Erythropoietin (EPO, Epoetin): stimulates RBC production. For anaemia in kidney failure. Produced in CHO cells. Tissue plasminogen activator (tPA, Alteplase): thrombolytic. CHO cells. For heart attacks and strokes. Interferon alpha/beta/gamma: antiviral, immunomodulatory. E. coli or CHO. Monoclonal antibodies (mAbs): Herceptin (trastuzumab, HER2+ breast cancer), Rituximab (CD20 lymphoma), Adalimumab (TNF-alpha, RA/Crohn). Produced in CHO or NS0 cells. Vaccines: Hepatitis B vaccine (HBsAg from yeast), HPV vaccine (VLPs from yeast/insect cells).

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9) has revolutionised transgenic animal production. How it works: guide RNA (gRNA) directs Cas9 nuclease to specific DNA sequence → Cas9 cuts both strands → cell repairs by: NHEJ (non-homologous end joining): error-prone → insertions/deletions → gene knockout. HDR (homology directed repair): precise editing if template provided → gene correction or knockin. Applications in transgenics: faster and cheaper than classical transgenic methods. More precise (fewer off-target effects). Can edit multiple genes simultaneously. CRISPR animals: pigs with human genes for xenotransplantation (PERV — porcine endogenous retrovirus knocked out + multiple human immune genes knocked in). Myostatin-knockout dogs (double-muscled, running ability). Disease model mice. CRISPR concerns: off-target edits, germline editing ethics, ecological risks of gene drives.