BIOL 311 Exam 1

cDNA cloning

Used to clone specific DNA inserts from larger eukaryotic genomes; lacks introns because it would inhibit expression; limitation: mRNA may not code for the DNA as we are starting with a small library of genes

northern blots

RNA and nucleotides; detection of specific RNA molecules among a mixture of RNA; used to analyze a sample of RNA from a particular tissue or cell type to measure the RNA expression of a particular gene

confocal microscopy

a light microscope that uses a laser, confocal pinhole, and images single points one by one; benefits are that it decreases stray fluorescence, optical section, stereo images, and multiple labels (several stains at once); two types

equilibrium density (rate zonal) centrifugation

a method for the separation of cells, organelles, macromolecules, or other particles of different densities by centrifugation in a solution that increases in solute concentration and hence in density

genomic cloning

a piece of DNA taken from the genome of a cell or animal then spliced into a bacteriophage or other cloning vector; can take a very long type if starting with the entire genome

radioactive tagging

a process in which a regular isotope of an element is replaced by a radioactive isotope of the same element in order to track the presence and concentration of that element in a biological sample

FRET biosensor

a reporter molecule that can indicate activity; protein kinase gets turned on with the help of ATP that was once ADP - can be reversible; when protein is activated it changes shape

scanning tunneling microscopy

a scanned proximity probe; instrument for imaging surfaces at the atomic level; DNA has a minor and major groove - first to image is the major

atomic force microscopy

a scanned proximity probe; no lenses (no abbe equation); revolutionary tip size; nuclear pores;

fluorescence immunocytochemistry

ability to identify the location of select proteins using antibodies; can achieve double label; can be direct or indirect; 1. fluorochrome 2. antigen - substance that elicits an immune response 3. epitope - part of the antigen where antibody binds 4. antibody - diverse but type used are bivalent 5. specificity (antibody mades binds to one specific antibody) vs affinity (tightness of binding)

Topic 16 - stem cells, tissue engineering, and regenerative medicine

adult stem cells - adipose (fat) derived used in more than 1000 stem cell therapy trials worldwide and includes adMSCs (adipose-derived mesenchymal stem cells); not as easy to produce but many sources (hair, teeth, etc), can be autologous match fetal stem cells - amniotic, umbilical cord, placental, but cannot use federal funds embryonic stem cells (hESCs) - now in clinical trials; easy to procure and more pluripotent (greater ability to differentiate) but not autologous match (self to self) induced pluripotent stem cells BioHeart repairing hearts through cellular cardiomyoplasty - famous autologous myoblast transplant -not FDA approved and cannot ship muscle overseas

knockout

an experiment in which a gene is deliberately inactivated in a living organism

transmission electron microscopy (TEM)

developed after bright field microscopy; use ultra thin sections of tissue; diffraction is limited; STEM is one type

densitometer

device that measures the density of a material; converts band densities to a bar graph that depicts the relative quantitative differences between bands; can be used in any gel electrophoresis

protease

enzyme that breaks down proteins; e.g. trypsin

cryostat

freezes tissues to make a hard block that cuts sections faster than traditional methods; dermatologists use it in Moh's surgery to treat skin cancers

knockin

gene of interest has been added to the genome or a native gene has been enhanced and expressed

knockout/knockin mice

genetically altered that assist researchers in understanding the genetic functions of the human body at greater level of detail; utilize cell cultures

molecular beacons

hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored when brought to a target nucleic acid sequence; nonradioactive method for detecting specific sequences and the presence of SNA; uses a quencher

MRI microscopy

has limited resolution; provides spatial and chemical information non-invasively; in vivo imaging

calmodulin biosensor

ionomycin induced activation of calmodulin (a calcium binding protein) then monitor the concentrations in and out of the cell to see if it worked; with addition of an ionomycin, calcium rushes into the cell and it lights up with fluorescence

forster resonance energy transfer (FRET)

ligand receptor interaction where binding of the ligand and receptor is reversible; works by pairing 2 fluorescent proteins together (CFP and YFP)

phase contrast microscope

light microscope that enhances contrast showing more detail; useful in examining living, unstained cells; does not use dyes

Topic 13 - government

president sets priorities; national institute of health (NIH) > national science foundation (NSF) > department of defense (DoD); NSF funds a lot of things including stem cell research; DoD funds new treatment for cancers; Trump says no more money to fund fetal stem cell research

scanning electron microscopy (SEM)

scanning the surface with a focused beam of electrons; electrons interact with atoms in the sample producing various signals that contain information about the surface topography and composition of the sample

Hayfilck's limit

showed that cell strains exhibit a finite number of doublings that inversely relate to the age of the donor

Topic 14 - stop/reverse aging

sirtuin genes (longevity genes) when over expressed in fruit flies increased max lifespan; each person has different stem cells; younger people have more; older people take longer for cells to differentiate and don't get as far as ours do

Topic 5 - labs (organs) on a chip

size of a thumb drive; mini organs on the same device; now being used for drug toxicity testing (in vitro toxicology) and blood brain barrier (BBB) and the effects of drugs on the brain and its blood vessels

3D printing (bioprinting)

technique to combine cells, growth factors, and biomaterials to fabricate biomedical parts that imitate natural tissue characteristics; can print tissues and organs to aid in research

patch clamp

technique used to study ionic currents in individual isolated living cells

biological scaffolds

structural support system; body's own cells replace it over time; there are biodegradable ones and permanent ones

mutations

the changing of the structure of a gene, resulting in a variant form that may be transmitted to subsequent generations, caused by the alteration of single base units in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes

differential interference contrast microscope (Nomarski)

looks at living cells; does not use dyes; has a 3D effect; used for single cell electrophysiology to get isolated neuron - need micropipette and to see cell surface

fluorescence recovery after photobleaching (FRAP)

measures some aspect of membrane fluidity by labeling a protein with a fluorochrome then photobleaching

veridex cell search system

method for detecting low number circulating tumor cells (CTCs); interaction of an iron labeled anti-EpCAM antibody to the surface of CTCs; FDA approved for CTCs

pulse-chase

method for examining a cellular process occurring over time by successfully exposing cells to a labeled compound (pulse) and then to the same compound in an unlabeled form (chase); useful for determining the activity of certain cells over a prolonged period of time; used to study protein kinase C, ubiquitin, and other proteins

affinity chromatography

method used to separate proteins based on ligand receptor interactions; best way to purify the insulin receptor which is on the outside of the cell

Topic 3 - Nanomedicine

nanoparticles to immuno-oncology; treating cancer with lasers - LANTERN targets and treats tumors; gold plated nanoparticles are shot into a tumor in a mouse which destroyed the tumor; nanomedical editing techniques - CRISPR cas9

laser-capture microdissection microscopy

one or two cells can be removed from a heterogeneous tissue section or group of cells for subsequent molecular analysis; isolates a single cell or cells from a tissue section for further analysis; uses laser ethylene-acetate film

ultrastructural tomography

used to obtain a 3D structure of sub-cellular macromolecular objects; uses TEM to collect data

trituration

used to process materials and to break open the cells using a syringe

photoreactive amino acids

uses UV light to make a covalent bond; put a western blot against it to track ligand-receptor interaction in living cells

deconvolution microscopy

uses an algorithm to create a 3D view of fluorescent stained cells; used to improve contrast and resolution

vivascope

uses confocal technology for imaging; less invasive technique than a skin biopsy

velocity sedimentation

uses gravity and does not use fluorochromes to sort; the larger ones go to the bottom and smaller ones take a longer time to go down

cell fixation process

1. fixation - fixed in place to look like its alive using formaldehyde 2. dehydration - swap water with ethanol 3. infiltration - swap ethanol with xylene then swap xylene with molten wax to make a bock of cells 4. Use microtome to cut sections 5. Use colorimetric stain to stain cells

electron microscopy

3 types that differ in the amount of light they produce; all pictures are black and white

CRISPR-Cas9

A gene-editing technique in which an endonuclease cuts genes identified by RNA "guide sequences" then the cell repairs the damage with an altered version

DNA gel electrophoresis

A method for separating DNA molecules by size; relies on restriction nucleases; uses polyacrylamide and agarose gels

differential centrifugation

A method used to separate the components of cells on the basis of their size and density

small interfacing RNA (siRNA)

A small, single-stranded RNA molecule generated by cellular machinery from a long, double-stranded RNA molecule; associates with one or more proteins in a complex that can degrade or prevent translation of an mRNA with a complementary sequence; short living

polymerase chain reaction (PCR)

A technique for amplifying and expanding the amount of DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides

southern blots

DNA and nucleotides; detection of specific DNA sequences in DNA samples; combines transfer of electrophoresis separated DNA fragments to a filter membrane; clinical applications - bone marrow transplant and first stem cell therapy

native gel electrophoresis

Protein molecules are attracted to a positively charged end of a gel and are sorted into bands based; smallest proteins move the farthest down the gel; preserves enzymatic activity of protein; resolution is not as good as SDS

dialysis

The diffusion of small solutes through a selectively permeable membrane; proteins remain in the bag

Topic 2 - Transplants without a waiting list

UNOS - united network for organ sharing; demand outpaces the supply but 3D tissue engineering can alter this

protein a immunoprecipitation

type of protein purification; commonly used as an antibody

Annexin V

used to detect apoptotic cells; turns green

propidium iodide

used to detect dead cells (via necrosis); turns red

band/gel shift assays

used to detect protein complexes with nucleic acids (DNA binding proteins); EMSA

enzyme-linked immunosorbent assay (ELISA)

a fluorescent technique (no microscopes) that is designed to quantitate a particular protein that is inside or outside of a cell; quantifies the relative amount of select proteins probed in multiwell plates using antibodies; all proteins added to the plate and like proteins will stick together; then add primary then secondary antibody; can be direct, indirect, ,or sandwich

complentation analysis

a test to see whether the mutations in two strains are in different genes; won't occur if they are in the same gene

total internal reflection fluorescence microscope (TIRF)

a thin region of specimen can be observed

guava

a type of flow cytometry, but uses microcapillary tubing for smaller samples; doesn't sort cells, but counts them

fetal bovine serum (FBS)

a type of supplemented medium that contains growth factors; type of animal serum that carries viruses making it not good for cell therapy; cell culture of cell strains

single nucleotide polymorphism (SNP)

a variation in a single base pair in a DNA sequence; research to predict symptom severity of autism spectrum disorder

Calcein-AM (acetoxy methyl ether)

a vital fluorescent dye; add AM to calcein makes it fluoresce; used to analyze cell membrane integrity

JC-1

a vital mitochondrial dye; if the control is orange/red and the experimental is green, then this means there is a difference in mitochondrial activity

Polarizing light microscopy

allows scientists to look at optical properties of the fiber using specific wavelengths of light; used by neurobiologists and muscle cell biologists

dynabeads/speedbeads

also microbeads that have an iron core; there is a magnet to pull them out of solution; you can add a selective surface

monocolonal antibodies

antibodies made by identical immune cells that are all clones of the parent cell; have monovalent affinity - they bind to the same part of the antigen that is recognized by the antibody; process of generating these was not patented but they are now among the top selling biologics in the world

ultrastructural immunocytochemistry

antibodies to tag proteins; gold labels to be lined to the Fc portion of the antibody probe; computer based 3D reconstruction

Topic 15 - bioterrorism, botulinum toxin and other nasty agents

bacteria that blocks the release of acetylcholine from nerve ends; interface RNA (RNAi) - double stranded DNA that blocks gene expression; has not yet been approved; Macular Degeneration - eye disease due to overproduction of blood vessels in the retina and too much production of vascular endothelial growth factor (VEGF); difficult for RNAi to get through the cell membrane; RNAi blocks the translation of proteins; ricin targets ribosomes which means that it kills by solely affecting protein synthesis and is not a neurotoxin

gel filtration

based on physical size and path length through a matrix; the big ones can't go through the gel so they come out first; the beads are porous

freeze fracture

breaks apart a frozen biological sample that can be examined in TEM; uses a platinum; can be used to view the interior of membrane (topology); can detect internal structure

micro spectrofluorometry/cytofluor

can quantify a fluorescent signal and express the data in a graphical form; used with cell culture; useful for analyzing vital fluorescent dyes

two photon microscopy

can reveal activities in tissues deeper than other alternative microscopes with less phototoxicity; uses live tissues

Topic 8 - cancer cells

cancer stem cells - low in number and don't respond to standard cancer treatments tumor microenvironment - cancer depends on the surrounding environment; extracellular signaling can keep cancer cells in non proliferating form or can trigger it genetically modified T cells - when injected into leukemia patients, it targeted and killed cancerous B cells inside the bone marrow conjugated antibodies - can bind only to cancer cells and then selectively kill them

growth substrate

cell culture plate; microcarrier beads; roller bottles - producing mAbs to replicate in vivo surrounds; microporous membrane; feeder layers - stem cells; neurospheres - balls of neural stem cells that differentiate

electroporation

commonly used to generate holes which small molecules can be delivered to the interior of the cell; puts an electrical current on either side of the cell; many cells die due to this injection technique

plastic thin sectioning

cutting specimens into extremely thin slices in preparation for TEM

lucifer yellow

designed for intracellular injection so that neurons can be traced in intact ganglia; needs to be a vital dye; can't be lipophilic

restriction nucleases

designed to cleave nucleotides at specific sites

fluorescence in situ hybridization (FISH)

detects the presence of genes in chromosomes and gene expression (mRNA); e.g. detects HPV in cancerous cervical HeLa cells

Topic 7 - Biomarkers

different forms including molecular marker (protein), circulating tumor cells, cell free DNA, exosomes or microvesicles that indicate the status of a possible or preexisting disease state sampled vis "liquid biopsy" (blood, urine, or cerebrospinal fluid); blood biopsies are very quick - only a few days and contain cfDNA from dead and recycled cancer cells; cell biomarkers - circulating tumor cells (CTCs); star trek tricorder x prize winners made a device that non-invasively detects changes that can indicate disease

transfection delivery methods

direct micro injection, biolistic particle delivery; electroporation; laser based transient transfection

green fluorescent protein (GFP)

discovered in jellyfish; a fluorescent protein whose DNA sequence is known; a reporter molecule when we want to track gene/protein expression in organisms and cells

Topic 9 - biotechnology

diverse set of disciplines designed to develop new medical devices, drugs, biomarkers, engineered cells, etc. for the purpose of improving human health and welfare; new cryosurgical device that super freezes target tumors such as pancreas; very expensive - value based pricing for drugs

fluorescence microscopy

excite at a particular wavelength and emits at a higher wavelength (produces heat); fluorochromes = fluorescent dyes

bright field microscopy

fixed, stained cells; used by pathologists

yellow fluorescent protein (YFP) and cerulean fluorescent protein (CFP)

fluorescent proteins used in FRET

flow cytometry - fluorescence activated cell sorting (FACS)

fluorochromes that can tag different cell types; uses fluorescent probes, lasers, and electrically charged plates

Topic 12 - religion, cell biology, and bioethics

human embryonic stem cells (hESCs) are derived from human embryos (blastocysts) and are now in clinical trials but requires the destruction of human embryos; Dickey-Wicker amendment bans the use of federal funding for this, but you can use private money; clones of Dolly the sheep born without lung disease and grew old; first successful cloning of primates (humans are primates)

Topic 4 - synthetic biology (Humulin)

humulin - comes from bacteria and used in synthetic biology/gene transfection; Dr. Craig Venter - construction of the first self-replicating synthetic bacteria cell and decoded the entire human genome in 2000; MITs nanobionics glow in the dark plant (2017) and improving photosynthesis (2019)

ultrastructural autoradiography

identifying dividing cells in breast tumors using 3H thymidine; can track cell processes over time; radioisotopes

cell line

immortal/unlimited number of cells; used to study cancer; origin in embryonic cells - 3T3 mouse origin

Topic 1 - Plants

improving human health with parsley and dill - GVA is extracted which inhibits the growth of tumor cells; pomegranates - prevents muscles form aging

viral transfection

intracellular injection technique using viruses

liposomes and nanoparticles

intracellular injection technique; microinjecting cells with a molecule of interest; bilayer synthetic carrier

high voltage electron microscopy

larger number and better resolution than TEM; best used for imaging thick sections

polycolonal antibodies

made using several different immune parent cells; use different epitopes; problem with specificity 1. purify antigen 2. inject into rabbit/mouse 3. B cells 4. divide - shed antibodies Problem - proteins are members of families with shared domain

cell strain

most in vivo like cells; cloned; mortal - Hayfiick's limit; immortal can be made by manipulating telomerase

cDNA microarrays

most powerful tool for studying gene expression in different organisms; described as a set of up to 8000+ spots that have either a red, green, yellow or no color; cDNA binds to complementary base pairs (hybridization)

spinning disk confocal microscopy

multiple lenses; multiple spots at once; decrease photo bleaching; decrease heat; good because can look at dynamic, moving changes in living cells within milliseconds (much faster)

necrosis

pathological cell death

2D gel electrophoresis

primary technique for proteomics; shows spots not bands; uses isoelectric focusing then SDS gel electrophoresis; separates the complex mixtures using two different properties of the proteins (PI value then molecular weight)

dark field microscopy

produces a bright image in a dark field; increases contrast but not resolution; special condenser; lens diffracts light; limited by the Abbe Equation; used by microbiologists

laser-scanning confocal microscopy

produces bright images; can use multiple stains; negative - takes a long time to image and not good for living cells (photobleaching and fluorochromes)

apoptosis

programmed cell death - cell knows it wants to die; how chemo works

western blot

proteins are identified by antibodies; qualitative (molecular weight) and quantitative (abundance)

SDS gel electrophoresis

proteins are pre-treated and are separated based on size; higher resolution 1. beta-mercaptoethanol - adds in H 2. urea - compromises hydrogen bead 3. SDS (negatively charged) and detergent 4. boil

single cell RNA-seq

provides the expression profiles of individual cells; patterns of gene expression can be identified through gene clustering analysis but its not possible to get complete info on every RNA expressed by each cell due to small amount of material available

Topic 11 - orphan diseases

rare diseases (<200,000 cases); spinal muscle atrophy (SMA) - leading genetic cause of death in infants and caused by deleted and defected genes that make proteins needed for proper muscle-nerve connections - Spinraza blocks the function of the defective gene and Zolgensma treats SMA by introducing the good gene through gene therapy fibrodysplasia ossificans progressive (FOP) - a gene that is supposed to be off but isn't which leads to muscles and tendons ossifying or turn to bone; its a fatal disease; no cure

microsomes

resealed membranes; rough - contains ribosomes; smooth - contains the plasma membrane and are where insulin receptors are found; rate zonal centrifugation separates the rough and smooth

DEAE and CM ion exchange chromatography

separated proteins based on ionic attraction to beads; typically requires NaCl gradient to elute the proteins

defined media

serum-free medium; all components defined; many cell types can be grown in defined medium; parabiotic - sharing the same blood system

small hairpin RNA (shRNA)

synthesized in the nucleus of transfected cells and form hairpin structures that consist of a stem region of paired antisense and sense strands connected by unpaired nucleotides that make up a loop; more challenging; can be permanent

transfection

the process of inserting genetic material such as DNA and double stranded RNA into cells; insertion allows expression and production of proteins

decellularization

the process used in biomedical engineering to isolate the extracellular matrix of tissue from its inhabiting cells - leaving the extracellular matrix scaffold of the original tissue

shRNA and siRNA

they target and degrade mRNAs and block the expression of a specific mRNA

microporous membranes

thin polymeric film or filament walled structures made up of microscopic pores; most critical in tissue engineered human skin; used as a filter

super resolution microscopy

three types (SIM, STED, and PALM); broke the Abbe's equation barrier - is not diffraction limited

EGTA

tissue dissociation into single cells; calcium chelator - takes calcium in tissue and absorbs it like a sponge but not a protease

Topic 10 - personalized medicine

treating patients based on their own unique genomic/proteomic profiles; development of technology to sequence your genome for under 1000 dollars; 23 and me started doing disease testing but FDA shut it down; genetic info nondiscrimination act of 2008 (GINA) - federal law that prevents discrimination of people based on their DNA or mutations

surface enhanced laser desorption ionization (SELDI)/matrix assisted laser desorption ionization (MALDI) - time of flight (TOF)

type of mass spectroscopy; uses a laser, TOF detectors and "protein chips"; resolution is one amino acid; biomarker discovery (e.g. Alzheimer's)

isoelectric focusing

tube gel; separates protein based on pH gradient; isoelectric point - pH where protein is neutral

tissue engineering

use of combination of cells; engineering material and suitable biochemical factors to improve or replace biological functions; e.g. human bladders; artificial livers (ELAD - filter block the infiltration of C3A cells so not FDA approved_; carticel knee cartilage; MatTek - human skin

vital fluorescent dyes

use of fluorochromes to monitor some change in physiological activity in living cells

autoradiography

use of radioisotopes to track cell changes; e.g. 3H thymidine - the cells that pick up this tracker will be the ones going through DNA synthesis; 3H leucine and 35S methionine are other examples; can be used to monitor protein synthesis as well

human embryonic stem cells (hESC)/stem cell therapy

use of stem cells to treat or precent a disease or condition

single cell microinjection using a micropipette

used for somatic cell nuclear transfer (SCNT), 3-parent baby, human-pig chimera, and tracing neurons using lucifer yellow

temperature sensitive mutants

variants of genes that allow normal functions of the organism at low temperatures but function alters at higher temps

Fluo3-AM

vital fluorescent dye; similar to cameleon

Topic 6 - Inherited Diseases

want to see if mitochondrial diseases can be prevented using nuclear transplantation and three parents; legal in UK not US