BIOL 216 - Topic 3

Different cell types result from

differential gene expression in cells with the same DNA

Differences between cells in a multicellular organism come almost entirely from

gene expression, not differences in the cell's genomes

When do differences between cells arise and how

they arise during development as regulatory mechanisms turn genes on and off

What is the intestine of C. Elegans derived from

the first four cells of the zygote

Genomic equivalence

nearly all cells of an organism having genomic equivalence

What occurs in nuclear transplantation

the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell, such as with Dolly the sheep

Can a transplanted nucleus support normal development of the egg

yes

What is the difference in outcomes between nuclear transplantation using a very differentiated cell versus a less differentiated cell as the nucleus donor in frogs

the transplantation performed using the less differentiated nucleus has a much higher probability of developing into a tadpole

Who is Dolly the sheep

in 1997 Scottish researchers were able to clone an adult sheep through nuclear transplantation from a differentiated mammary (udder) cell that was de-differentiated, there were 277 attempts to fuse the adult cells with enucleated eggs, and Dolly was the only one to survive and be born as a lamb

How are the cells de-differentiated

they are deprived of nutrients in order to arrest the cell cycle

What ended up happening to Dolly

she grew to adulthood and produced offspring, but she died prematurely and has arthritis which led to thinking that her cells were older than a normal sheep of the same age which may have been due to incomplete reprogramming of the nucleus that was transplanted originally

What are naturally occurring clones

identical twins, can have different characteristics

Main issue associated with animal cloning

only a very small percentage of the clone embryos are able to develop normally to birth

What are cloned mice prone to

obesity, pneumonia, liver failure, premature death

Why does cloning occur with such low rates of efficiency

in order for genes to be expressed or repressed appropriately in early stages of development the many epigenetic changes (such as histone acetylation or DNA methylation) need to be reversed from the donor nucleus

What factors and processes affect epigenetic mechanisms

development (in utero, childhood), environmental chemicals, drugs/pharmaceuticals, aging, diet

DNA methylation

methyl group (an epigenetic factor found in come dietary sources) can tag DNA and activate or repress genes

Histones

proteins around which DNA can wind for compaction and gene regulation

Histone modification

the binding of epigenetic factors to histone "tails" alters the extent to which DNA is wrapped around histones and the availability of genes in the DNA to be activated, is a post-translational modification, includes: methylation, phosphorylation, acetylation, and ubiquitylation

Epigenetic mechanisms include

DNA methylation and histone modification which alters how genes are expressed without altering the underlying DNA sequence

Stem cell

a relatively unspecified cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types

Three types of stem cells

totipotent, pluripotent, multipotent

Totipotent

ability to give rise to every type of cell in the adult body, ex: zygote

Pluripotent

can form many different cell types, ex: embryonic stem cells

Embryonic stem cells

ESC, cells taken from the inner cell mass of the blastocyst, give rise to all three types of germ layer cells

Multipotent

ability to differentiate is limited to a few cell types, ex: bone marrow cells, blood within the umbilical cord containing undifferentiated cells (can be used for bone marrow transplants)

Benefits of therapeutic cloning

the donor nucleus comes from a cell of the person that is receiving the ES cells meaning that they won't be rejected by the immune system, can keep the cultured pluripotent stem cell undifferentiated by adding chemicals to media

Cell types in the ectoderm

skin cells, neurons, pigment cells

Cell types in the mesoderm

cardiac, smooth, and skeletal muscle, tubule cell of kidney, red blood cells

Cell types in the endoderm

lung cell, thyroid cell, pancreatic cell

Induced pluripotent stem cells (iPSCs)

adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of ESCs

How were differentiated cells transformed into ESCs

by introducing them to extra copies of 4 "master regulatory" genes

Retrovirus

RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome, can be "packaged" for "gene delivery", ex: HIV

What happens to the DNA from a retrovirus

it's incorporated into the host's genome by an integrase enzyme

What method does the coronavirus utilize to infect cells

RNA-dependent RNA polymerase, not reverse transcriptase

Reverse transcriptase

an enzyme used to generate complementary DNA from an RNA template

Viral multiplication steps of an RNA virus

1. Adsorption, 2. Penetration, 3. Uncoated, 4. Synthesis: replication and protein production, 5. Assembly, 6. Release

Adsorption

the virus attaches to its host cell by specific binding of its spikes to cell receptors

Penetration

the virus is engulfed into a vesicle and its envelope is uncoated

Uncoated

thereby freeing the viral RNA into the cell cytoplasm

Synthesis: replication and protein production

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

Assembly

viral spike proteins are inserted into the cell membrane for the viral envelope; nucleocapsid is formed from RNA and capsomers

Release

enveloped viruses bud off of the membrane, carrying away an envelope with the spikes, this complete virus or virion is ready to infect another cell

Lentiviral vector

a tool used by molecular biologists to deliver genetic material into cells

How do lentiviral vectors work

delete the genes that make the virus cause disease, leave the genes that allow the virus to package and deliver the desired genetic material into the cell

iPS cells were first created in 2006 by the forced expression of

four "reprogramming" transcription factors known as the Yamanaka reprogramming factors, utilizing retroviruses for delivery

Yamanaka reprogramming factors

Oct4, Sox2, c-Myc, and Klf4

Issues with iPS cells

still differences between them and ES cells such as DNA methylation errors

Caveats of using retroviruses

with some retroviral vectors random insertion into the genome can cause mutations that can lead to cancer

Induction of pluripotent stem cells summary

1. Somatic cells are obtained from the adult organism, 2. The reprogramming factors are introduced into the cultured somatic cells, 3. The cells are grown under embryonic stem cell conditions and after 2-3 weeks the iPS cells emerge, 4. These induced pluripotent stem cells may be differentiated into various cell types for regenerative medicine applications

Benefits of stem cells

ESC can theoretically give rise to all the types of cells in an organism, can be used to repair a damaged or diseased organ

What can bone marrow stem cells do

generate different types of blood cells, can differentiate into bone, cartilage, fat, and muscle

Stem cells can help fix

Type 1 diabetes, Parkinson's, Huntington's, Alzheimer's, damaged immune systems (due to autoimmune disorders or radiation treatment for cancer), spinal cord injury, and burns/skin grafts

Stem cells use for Type 1 diabetes

insulin producing pancreatic cells

Stem cells use for Parkinson's

add/replace dopamine producing cells

Stem cells use for Huntington's

add/replace neurons that produce GABA

Stem cells use for Alzheimer's

add or replace neurons that produce ACh

Gene therapy steps

1. Insert RNA version of normal allele into retrovirus, 2. Let retrovirus infect bone marrow cells that have been removed from the patient and cultured, 3. Viral DNA carrying the normal allele inserts into the chromosome, 4. Inject engineered cells into patient

Who was the first person to die in a clinical trial for gene therapy

Jesse Gelsinger

What did Jesse Gelsinger have

ornithine transcarbamylase deficiency

Ornithine transcarbamylase deficiency

an X-linked genetic disease of the liver characterized by an inability to metabolize ammonia (a byproduct of protein breakdown)

Why did Jesse Gelsinger die

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 by the use of the viral vector used to transport the gene into his cells which led to multiple organ failure and brain death

SCNT

somatic cell nuclear transfer, ex: Dolly the sheep

CRISPR stands for

clustered regularly interspersed short palindromic repeats

CRISPR

segments of prokaryotic DNA containing short repetitions of base sequences that are interspaced with sequences derived from viruses with each repetition being followed by a spacer DNA

Cas9

"CRISPR associated 9" nuclease

What is CRISPR originally

an immune defence system found in bacteria and archaea that results in the degradation of invading DNA

CRISPR is utilized as a

tool to target locations in complex mammalian genomes and generate site specific breaks in DNA

The CRISPR system can be modified for

knock out of a target gene, knock in (insertion of a donor template into a target region), targeting the promoter region of the gene to upregulate or downregulate transcriptional activity

What can a "knock in" of genes accomplish

"fix" a mutation, introduce a tag, create a new restriction site

How does CRISPR-Cas9 work

it delivers the Cas9 nuclease complex with the appropriate guiding RNAs into a cell fo that the DNA can be cut at the targeted region

Important parts of the CRISPR-Cas9 system

Cas9 nuclease, sgRNA, crRNA, tracrRNA, PAM

sgRNA

single guide RNA

crRNA

target specific CRISPR RNA, binds to targeted DNA

tracrRNA

trans-activating crRNA, base pairs with crRNA and enables the Cas9-crRNA complex to locate the targeted DNA, is necessary to activate the enzymatic function of Cas9

PAM

protospacer-adjacent motif, RNA sequence following the target DNA sequence, necessary for Cas9 to bind and cleave the target DNA

Steps of CRISPR

1. Guide RNA binds to target sequence, 2. Cas9 enzyme binds to guide RNA, 3. Cas9 enzyme cuts both strands of DNA, 4. The cut is repaired inducing mutation

Double stranded breaks made by Cas9 are repaired by two types of mechanisms

NHEJ and HR

NHEJ

non-homologous end joining, DNA ends are ligated back together, usually with the introduction of a small insertion or deletion

HR

homology directed repair, donor DNA with homologous sequences at either end can be integrated into the site

Cell fate can be manipulated by

activating specific endogenous gene expression with CRISPR-mediated activator

What modification has been made to dCas9

no longer cuts DNA but still can be guided to and bind to specific sequences

dCas9 combined with transcription activators

can manipulate endogenous gene expression

What did Wei et al do

enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators and directly converted mouse embryonic stem cells into two extraembryonic lineages