Topic 3: Cell Determination and Stem Cells

For Quiz 3. Part 1: cloning and embryonic stem cells (ESCs). Investigates the connection between unspecified stem cells and cell specialization.

Genomic Equivalence

The theory that all cells of an organism contain the same genetic information.

Differences between the suggests that cells express their DNA differently via differentiation.

Totipotent Cell

A single cell that can technically give rise to a new organism, given appropriate maternal support.

Cellular Enucleation

The removal of a cell's nucleus, creating a nucleus-free cell (cytoplast) often by exposing the cell to UV rays.

Nuclear Transplantation of Less Differentiated vs. More Differentiated Frog Cells

An enucleated egg cell received one of two donor nuclei via transplantation.

The first donor came from a frog embryo of a less differentiated cell. It was then activated to begin development, with most cells developed into tadpoles.

The second donor came from a frog tadpole’s fully differentiated intestinal cell. When activated to begin development, most celled stopped before reaching the tadpole stage.

Implications of Frog Nuclear Transplantation

Specialized cells loose their ability to “revert” back to totipotency. There is something in the cells of the embryo that still enable them to give rise to all the cells in the organism, but this ability seems to be lost as development continues.

Somatic Cell Nuclear Transfer (SNT/SCNT)

A cloning technique where a somatic cell nucleus is transferred into an enucleated oocyte, creating a genetically identical clone of the DNA donor.

Ex. Dolly the sheep was lab-grown into an embryo after receiving a donor egg cell from one sheep, donor mammary cells from another sheep (whom she was genetically identical to), and implanted into the uterus of a third sheep. Dolly was the first out of 277 SCNT attempts to survive.

Scientists used an organism’s cells to create totipotent (embryonic-like) tissues.

Problems Associated with Somatic Cell Nuclear Transfer (SCNT)

Only a small percentage of cloned embryos, like Snuppy the dog, have developed normally until birth.

Dolly died prematurely with arthritis, suggesting that her cells were “older” due to incomplete reprogramming of the original transplanted nucleus.

Mice are prone to obesity, pneumonia, liver failure, and premature death.

Why Are There Problems with Cloning?

Epigenetics. Cloned animals may look or behave differently due to environmental and random events during development.

We cannot control and replicate the exact epigenetic landscape that prevents histones from modifying specialized cells.

Histones

Proteins found in chromosomes that bind to DNA, helping to give chromosomes their shape. Controls the activity and expression of genes via their positively-charged tails.

Incomplete Epigenetic Reprogramming

DNA in cloned embryos show more methyl groups than DNA from normal embryos. Differentiated cells have more methylation.

Epigenetic changes must be completely reversed in the nucleus from a donor animal for genes to be expressed/repressed appropriately for early stages of development.

Histone (not DNA) Methylation

A reversible epigenetic process adding methyl groups to histone tails to serve as docking sites for other proteins, altering long-term gene expression (blocking/activating promoters or transcription factors) and protein production.

Does not change the charge on histone tails; rather creates new binding sites for specific proteins.

Histone Modification

A covalent post-translational modification (PTM) to histone proteins. In other words, small chemical groups are covalently (strongly) attached, often on histone tails.

• Acetylation adds an acetyl group (–COCH₃)

• Methylation adds one or more methyl groups (–CH₃)

• Phosphorylation adds a phosphate group (–PO₄³⁻)

• Ubiquitination adds the protein ubiquitin

Acetylation

A reversible epigenetic process that introduces an acetyl group to lysine-rich N-terminal histone tails, turning the charges from positive to neutral and loosening their grip on DNA. Upregulates gene expression.

Deacetylation

A reversible epigenetic process that removes acetyl groups from lysine-rich N-terminal histone tails, turning the charges from neutral to positive and tightening their grip on DNA. Downregulates gene expression.

Phosphorylation

A reversible addition of a short-term negatively charged phosphate group to histone tails, turning the charges much more positive and loosening their grip on DNA. Upregulates gene expression.

Ubiquitylation/Ubiquitination

A reversible addition of ubiquitin protein to histone tails to serve as short-term docking sites for other proteins to access chromatin, altering gene expression (blocking/activating promoters or transcription factors) and protein production.

Does not change the charge on histone tails; rather creates new binding sites for specific proteins.

Stem Cell

A relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types.

Embryonic Stem Cells (ESC)

Cells taken from the inner cell mass (ICM) of the blastocyst. Can theoretically give rise to all the types of cells in an organism.

Bone Marrow Stem Cells

Immature, specialized cells located in the marrow that produce all blood components: red cells, white cells, and platelets, as well as bone, cartilage, fat, and muscle.

Pluripotent Cell

A cell that is typically taken from an ICM (epiblast) that can form all different body cell types. Limited because they already differentiated once and cannot form extraembryonic cells.

Ex: ESC

Multipotent Cell

A cell whose ability to differentiate is limited to a few cell types, depending on their location in the embryo and surrounding cell signaling.

Why are Pluripotent Embryonic Stem Cells Sought After?

They have the unique ability to develop into any cell type in the body, making them a potential source for regenerating damage.

Offers a treatment option for a wide range of diseases involving complex tissues/structures that are not viable for transplant, and lowers the change of rejecting transplanted tissue.

Therapeutic Cloning

A technique that uses nuclear transfer of a somatic cell (SCNT) to generate embryonic stem cells (ESCs).

Since the donor DNA matches the recipient DNA of ESCs, immune rejection of the tissue is less likely.

Induced Pluripotent Stem Cells (iPS/iPSCs)

Adult cells (ex. skin) that have been genetically reprogrammed (induced) to an embryonic stem cell–like state. They are forced to express genes and factors important for maintaining properties of embryonic stem cells.

Differentiated cells are induced through the use of retroviruses and 4 “master regulatory” genes.

Insertion of iPS is believed to be carcinogenic and can lead to mass organ failure.

Yamanaka Reprogramming Factors

Transcription factors created from the 4 “master regulatory” genes and delivered via engineered retroviruses grown under lab conditions.

They mimic the condition of early cells.

Oct4, Sox2, c-Myc, and Klf4

c-Myc shuts down specialization while others turn on pluripotency. Thought to be carcinogenic.

Capsomer

A subunit of a capsid, which is an outer protein coating that protects viral genetic material.

Virus

A non-living agent that infects another cell via hijacking a cell’s genome to edit it and create copies of itself.

The virus does this by attaching itself to a host cell via binding spikes to cell receptors. It is then engulfed into a vesicle and its envelope is uncoated, freeing the viral RNA into the cell cytoplasm.

Under the control of viral genes, the cell synthesizes basic components of new viruses: RNA molecules, capsomers, and spikes.

Viral spike proteins are inserted into the viral envelope while nucleocapsid is formed from RNA and capsomers. Enveloped viruses bud off of the membrane or lyse the cell, ready to infect another.

Retrovirus

An RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme.

Retroviruses can be “packaged” for “gene delivery” (we can transform retroviruses from something harmful into tools that deliver engineered genetic material into a target cell's genome).

Lentiviral Vectors

A tool used to deliver genetic material into cells.

Humans have found a way to delete the genes that make the virus cause disease and leave the genes that allow packaging and delivery of desired genetic material.

Are iPSCs and ESCs Identical?

No. iPSCs reprogrammed adult cells; ESCs come from embryos. DNA methylation errors will differ.

Gene Therapy

A technique that treats or prevents diseases by correcting, replacing, or editing (like adding/removing) genes. Encompases ESCs, SCNTs, and iPSCs. You can do this by:

Inserting the RNA version (from a cloned gene) of the normal allele into the retrovirus. Then let the retrovirus infect bone marrow cells that have been removed from the patient and cultured.

Viral DNA carrying the normal allele inserts itself into the chromosome. The infected normal-allele bone marrow cells are injected back into the patient’s own bone marrow.

Ornithine Transcarbamylase (OTC) Deficiency

An X-linked genetic disease of the liver that lacks the ability to create an enzyme that metabolize ammonia (a toxic byproduct of protein breakdown). Without OTC, the body builds up orotic acid.

Jesse Gelsinger

The first person who died in a clinical trial for gene therapy. He suffered from ornithine transcarbamylase deficiency and was injected with an adenoviral vector carrying a corrected gene to test the safety of the procedure.

He died four days later having suffered a massive immune response, triggered using the viral vector used to transport the gene into his cells. This led to multiple organ failure and brain death.

CRISPR-Cas9

An immune defense system found in bacteria & archaea. Operates with the goal of degrading invading viral DNA.

Utilized as a tool to target locations in complex mammalian genomes and generate site specific breaks in DNA from modification.

• Knock out a target gene

• Knock in a donor template into the target region (“fix” a mutation, introduce a tag, create a new restriction site)

• Target the promoter region of a gene to upregulate or downregulate transcriptional activity.

Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) Locus

Segments of prokaryotic DNA containing short repetitions of base sequences, interspaced with sequences derived from viruses.

Composed of a pattern of repetition and a corresponding spacer DNA.

Protospacers

Small pieces of viral DNA processed by Cas1 and Cas2 (adaptation complex) before it is stored into the bacteria’s CRISPR gene.

Spacers

Viral agents permanently stored in the bacterial CRISPR locus.

CRISPR- Cas9 Immune Response: Acquisition

The invading phage DNA is processed by the adaptation complex into small DNA fragments, called protospacer sequences, and then incorporated into the CRISPR locus of the bacterial genome as a new spacer.

pre-crRNA

A long RNA produced when the host bacterium transcribes the CRISPR locus. Looks like this:

spacer RNA - repeat RNA - spacer RNA - repeat RNA

One of the “scissor handles” that is involved in hybridization.

Mature crRNA/CRISPR RNA

Processed pre-crRNA where the multiple consecutive digits of spacers and their repeats are cut down to one each.

Looks like this:

spacer RNA - partial repeat RNA

The functional “scissor handles.”

Trans-Activating crRNA (tracrRNA)

RNA that has a sequence complementary to the repeat section of pre-crRNA and mature crRNA. Capable of hybridization, which base-pairs to repeat crRNA regions and turning those regions into double-stranded DNA.

One of the “scissor handles” that is involved in hybridization.

Endogenous

Originating from within an organism.

Endogenous Gene

A gene that is naturally present within an organism's genome.

RNase III

A bacterial host’s ribonuclease that recognizes double-stranded RNA and cuts the repeat regions bound to tracrRNA. What’s left is a spacer and a part of the repeat region, called the mature crRNA. tracrRNA is still attached.

sgRNA/Single Guide RNA

Umbrella term that includes target specific crRNA, CRISPR RNA, and tracrRNA.

CRISPR- Cas9 Immune Response: Biogenesis

The CRISPR locus is transcribed into pre-crRNA.

tracrRNA is transcribed from the tracr gene, upstream of the CRISPR gene locus. Has a sequence complementary to the repeat regions of the CRISPR gene locus.

TracrRNA hybridizes to the repeat sequences of the pre-crRNA.

Endogenous RNase III cleaves this complex, yielding mature crRNAs with tracrRNA still attached, each containing one spacer and a partial repeat sequence.

Cas9/CRISPR Associated 9 Nuclease

A DNA cutting enzyme, directed by CRISPR RNA. The “scissor blades.”

PAM Protospacer-Adjacent Motif

A DNA sequence following the target DNA sequence. Necessary for Cas9 to bind and cleave the target DNA.

CRISPR- Cas9 Immune Response: Interference

Mature crRNA with tracrRNA attached guides Cas9 protein (working scissors) to the complementary foreign nucleic acids and PAM, triggering degradation of the DNA sequences of invading phages.

• The cut is repaired by introducing a mutation in the DNA.

• Enzymes are engineered to remove and rejoin DNA.

• DNA repair system can be hijacked to inset a section of DNA into the genome.

Non-Homologous End Joining (NHEJ)

A common repair mechanism after a double strand break caused by Cas9 that ligates DNA ends indiscriminately back together, but usually with the introduction of small indels at the repair site. Active throughout the entire cell cycle. Operates with no chromosome template and thus ignores the original sequence.

Quicker but less accurate. Prioritizes large knock-outs.

Homology Directed Repair (HDR)

A repair mechanism after a double strand break caused by Cas9 that recognizing homologous sequences of DNA (from a sister chromatid, a donor homology plasmid, single stranded ODN, etc.) and uses those homologous regions as a template for precise damage correction.

Precision is much higher here due to referral to homologous template. Less efficient as it is only active during late S and G2 phases of the cell cycle.

Dead Cas9 (dCas9)

A modified CRISPR-Cas9 protein that can't cut DNA but still precisely binds to specific DNA sequences guided by guide RNA.

Combined with transcription activators, it can manipulate endogenous gene expression by staining/marking them.