Lecture 1 – Study of Cells: Genetic Manipulation and Analysis

Vocabulary flashcards generated from Lecture 1 covering cell culture types, immortalization mechanisms, gene transfection, CRISPR-Cas9 genome editing, nucleic acid detection, and computational cell biology tools.

In vitro

Refers to cell culture procedures and biological processes conducted outside a living organism, typically on plastic or glass culture vessels.

In vivo

Refers to biological processes occurring within a fully intact, living organism where cells interact inside their natural tissue context.

Ex vivo

Refers to cells, tissues, or organs harvested directly from an in vivo living source and subsequently maintained or analyzed in an in vitro culture environment.

Hayflick limit

The finite threshold of approximately 40 to 60 population doublings that normal human cells can undergo before entering permanent replicative senescence due to progressive telomere shortening.

Senescence-associated secretory phenotype (SASP)

The state of senescent cells characterized by an enlarged, flattened morphology, loss of proliferative capacity, elevated expression of the tumor suppressor $p16^{INK4a}$, and increased availability of senescence-associated $\beta$-galactosidase.

EGTA

Ethylene glycol-bis($\beta$-aminoethyl ether)-N,N,N',N'-tetraacetic acid; an aminopolycarboxylic acid chelating agent preferential to $Ca^{2+}$, used to disrupt cell-cell and cell-ECM junctions during primary cell culture isolation.

Immortalized cells

Cultured cells engineered (e.g., via TERT expression) to maintain telomere length, providing unlimited replicative capacity while typically preserving cell-cycle checkpoint control and karyotypic stability.

Transformed cells

Cultured cells expressing an oncogene or lacking tumor suppressor genes (e.g., $p53$ loss) that have lost cell-cycle checkpoint control, resulting in continuous growth, chromosomal instability, and accumulated DNA damage.

TERT

Telomerase reverse transcriptase; the catalytic enzymatic DNA polymerase subunit of telomerase that uses an RNA template to synthesize telomeric repeats at the 3' ends of chromosome lagging strands.

TERC

Telomerase RNA component (also termed TR); the non-coding RNA component of the telomerase complex that provides the template sequence for extending telomeric DNA.

Organoid

A 3D organ-like structure grown in vitro that mimics the native cellular architecture, matrix signaling, and gene expression patterns of tissue in vivo.

Adenoviral vector

A non-enveloped dsDNA viral vector system that enters target host cells via the CAR and integrin co-receptors to deliver high-level, transient gene expression without integrating into the host genome.

Lentiviral vector

A subclass of retrovirus containing an ssRNA genome that reverse-transcribes into DNA and integrates into host genomes, enabling persistent gene expression in both dividing and non-dividing cells.

Electroporation

A physical transfection method where an applied electrical field exceeding the cell membrane voltage threshold creates transient hydrophilic pores in the plasma membrane to permit entry of nucleic acids.

Biolistic particle delivery

A physical transfection technique (particle gun) in which nucleic acid-coated gold or tungsten microparticles are propelled at high velocity into recipient cells.

pCMV6-XL5

An example mammalian expression vector containing an SV40 origin for mammalian replication, a CMV promoter for strong expression, a multiple cloning site (MCS), a PolyA signal, T7/M13 promoters, a ColE1 bacterial origin, and an ampicillin resistance gene.

Luciferase

An oxidative reporter enzyme derived from bioluminescent organisms that emits light upon catalyzing the oxidation of its substrate luciferin.

Green Fluorescent Protein (GFP)

A fluorescent marker protein composed of an 11-strand $\beta$-barrel enclosing a tripeptide chromophore (Ser65, Tyr66, Gly67) that emits green fluorescence under blue light illumination.

Casgevy (exagamglogene autotemcel)

The first FDA-approved CRISPR-Cas9 somatic cell therapy (approved December 2023), which edits a patient's hematopoietic stem cells ex vivo to boost fetal hemoglobin production for treating sickle cell disease.

Protospacer adjacent motif (PAM)

A short 2 to 6 base pair DNA sequence (e.g., 5'-NGG-3' for SpCas9) immediately adjacent to the targeted genomic locus, required for Cas nuclease recognition and cleavage.

crRNA

CRISPR RNA containing a 17 to 20 nucleotide sequence reverse-complementary to the target genomic sequence of interest.

tracrRNA

Trans-activating crRNA; the non-variable RNA structural element in the CRISPR complex that binds directly to the Cas nuclease protein.

Non-Homologous End Joining (NHEJ)

An error-prone double-strand DNA repair pathway that joins broken ends without a template, frequently creating insertion or deletion (indel) mutations that cause target gene disruption (knock-out).

Homology-Directed Repair (HDR)

A precise double-strand DNA repair pathway that uses a homologous repair template sequence to introduce specific gene modifications, sequence corrections, or transgene insertions.

Fluorescence In Situ Hybridization (FISH)

A cytogenetic technique using fluorescently labeled nucleic acid probes to locate complementary RNA or DNA sequences within preserved tissues, cells, or metaphase chromosomes.

Rolling Circle Amplification (RCA)

An isothermal enzymatic DNA amplification technique utilized in spatial transcriptomics platforms (e.g., 10X Genomics Xenium) to locally amplify ligated padlock probes for single-transcript optical detection.

SYBR Green

A fluorescent dye that intercalates specifically into double-stranded DNA to quantify PCR products in real-time quantitative PCR assays.

Taqman probe

An oligonucleotide hybridization probe labeled with a fluorescent reporter and a quencher; sequence-specific binding and subsequent 5'-to-3' exonuclease cleavage during PCR extension releases the reporter signal.

Cellpose

A deep-learning AI segmentation algorithm designed to automatically segment individual cells and nuclei across varied imaging modalities without requiring manual re-tuning.

DeepCRISPR

A deep-learning computational model used to evaluate and predict both the on-target cleavage efficiency and off-target cleavage risk of designed CRISPR guide RNAs.