Mcb 2050 Topic 3

CAG, Polyglutamine

3-base pair deletion of ctt causing ∆F508

transgene

deoxyadenosine

protein folding

Mosaics

chimeras

Tumor gene (T-region)

mutating it

random integration

Gain of function mutation, trinucleotide repeat disease

CAG repeated >42 repeats results in a polyQ tract causing proteins to misfold forming toxic aggregates killing neuron cells. Normal cells have <35 repeats. CAG codes for glutamine (Q).

Huntingtin's gene= HTT

Diagnosis by PCR to determine size of region containing CAG repeats

loss of function mutation in the cystic fibrosis transmembrane conductance regulator CFTR gene

codes for an ion (chloride) channel that regulated water balance in epithelial cells

ctt deletion deleting a phenylalanine in codon 508. Called ∆F508. Causes CFTR protein to misfold within the endoplasmic reticulum resulting in its degradation

Diagnosis by Southern blotting of PCR products using allele-specific probes.

With positional cloning techniques

Correct a mutant phenotype by adding a normal copy of a gene (transgene) to the genome of an individual carrying a defective copy

Recombinant retroviruses allow stable transgene insertion into the genome

The insertion site is random

adenosine deaminase-deficient severe combine immunodeficiency disease due to accumulation of deoxyadenosine in absence of ADA activity. Deoxyadenosine is toxic to immature lymphocytes causing premature death and immune deficiency

bone marrow transplant

gene therapy inserting transgenic lymphocytes

mutations in the IL2Rγ2 gene results in death of T cells due to lack of interleukin-2

1.wild-type gene inserted into retroviral vector. 2. Used to infect patient derived bone marrow stem cells.

3.Grow in culture and implanted back into patient

recombinant plasmids used to express eukaryotic proteins of economic value in genetically engineered bacteria or eukaryotic cells.

insulin, growth hormone, clotting factor IV)

pro: cost effective

con: lack eukaryotic post-translational modifications

yeast, insect and mammalian cell lines, and transgenic plants and animals

1. remove eggs from female mouse and fertilize in vitro

2. microinjections of DNA vector containing transgene

3. implantation into pregnant mouse

4. Screen off spring for transgenic mice

The offspring of eggs from female mouse whose eggs had microinjections DNA vectors. DNA integrates at random making them mosaics

(i) mate the black mice and remove blastocyst from pregnant female

(ii) isolate embryonic stem cells (ES) tom the blastocyst and culture these vito

(iii) transfect transgene into ES cells

(iv) inject transfected ES cells into freshly isolated blastocysts from light coloured parents

(v) implant blastocysts into female white mice

embryonic stem cells. Pluripotent stem cells derived from the inner cell mass of blastocyst

Simplified by transfection of transgene into black donor ES cells that are injected into white blastocytes- chimeric offspring are mated to produce black offspring that are potentially transgenic

Progenies of pseudopregnant females. have two types of cells that have contributed to the formation of adult tissues

using recombinant Ti plasmids

Ti plasmid of Agrobacterium tumefaciens - bacterium infects plants. T-DNA region of Ti plasmid replaces tumor genes with transgene and required for plant cell transformation

Recombinant Ti plasmid replaces tumor genes with transgene and includes antibiotic resistance genes.

Plasmid is inserted into A. tumefaciens, which are used to infect plant cells that are regenerated into transgenic plants in vitro

express mutant EPSP synthase that is resistant to the herbicide glyphosate

targeting vector containing neomycin resistance gene inserted such that it disrupts the coding sequence of a gene of interest.

Homologous recombination replaces the genomic copy with the disrupted copy.

Allows selection against cells that have integrated the vector randomly in the genome (not by homologous recombination) These cells will be killed by gancyclovir (tocis nucleotide analog incorporated into DNA with phosphorylated by thymide kinase)

killed by neomycin

killed by gancyclovir

survive

collection of transgenic plants with genes disrupted due ti random T-DNA insertion

dsRNA or short hairpin RNA can bind to mRNA 3'UTR and inhibit translation or cause mRNA degradation

Uses same pathway that cells naturally use to silence gene expression (miRNA/RISC) This technique is used to silence the expression of any gene for which the gene sequence is known

exploits a natural defence system used by bacteria to guard against bacteriophage infection.

1. A single guide RNA (sgRNA) targeting a gene of interest is transfected into cells together with the gene encoding the Cas 9 nuclease

2. A double-strand cut is made within the genome at the target site, which is repaired by the cellular DNA repair enzyme

3. Non-homologous end joining repair leaves insertions/deletions causing gene inactivation.