Fundamental Cellular Processes

• Generation of Transgenic Livestock and Rodents:
• Pronuclear DNA Microinjection (Gordon & Ruddle, 1980):
• Linearized recombinant transgene constructs are injected directly into the male pronucleus of freshly fertilized embryos at the One-Cell Stage (zygote).
• Performing microinjection at the single-cell stage prior to the first mitotic cleavage ensures that the integrated transgene is replicated and passed into every embryonic cell lineage, minimizing germline mosaicism.
• nalysis:
• (One-cell-stage): Correct developmental stage.

Correct option: (1) One-cell-stage.

• CAR-T Cell Therapy Approval in India:
• In October 2023, India's Central Drugs Standard Control Organisation (CDSCO) approved NexCAR19 (actalycabtagene autoleucel), India's first indigenous chimeric antigen receptor (CAR) T-cell therapy developed by IIT Bombay and ImmunoACT.
• Clinical Indication: Approved for patients with relapsed or refractory B-cell malignancies, specifically B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL), by genetically engineering patient T cells to express a CAR targeting the CD19 surface antigen.

Correct option: (2) Refractory B-cell lymphomas and leukemia.

• HAT Medium Selection Mechanism:
• HAT medium contains Hypoxanthine, Aminopterin, and Thymidine:
• Aminopterin: A potent folate analog that blocks dihydrofolate reductase (DHFR), shutting down de novo synthesis of purines and thymidylate.
• Hypoxanthine & Thymidine: Allow cells to survive via salvage pathways if they possess functional salvage enzymes:
• HGPRT (Hypoxanthine-guanine phosphoribosyltransferase) for purine salvage.
• TK (Thymidine kinase) for pyrimidine salvage.
• In hybridoma generation, parent myeloma cells deficient in HGPRT (or TK) die in HAT medium. Primary spleen cells cannot divide indefinitely in culture. Only hybrid cells, which inherit functional HGPRT/TK from B cells and immortality from myelomas, survive.

Correct option: (1) Thymidine kinase and Hypoxanthine guanosine phosphoribosyl transferase