Week 3 Flashcards
(19 cards)
Epigenetics
Study of GE/phenotype changes not caused by DNA sequence changes
> Mendelian: reciprocal cross @ autosomal locus –> same phenotype despite parent allele origin/sex
Genomic Imprinting
- Mom influence first: heritable and reversible
2. 50-200 gene loci; fetal growth; cell proliferation
DNA methylation
Cytosine at CpG island; DNA, S-adenosylmethionine and DNA methyltransferase needed
1. De Novo: unmarked with DNMT3A and T3B
2. Maintenance: marked by methylation –> replication –> dsDNA hemimethylated twice –> DNMT1 and VHRF1 methylate both
> RESULTS: H3 deacetylation and methylation at lysine 9 –> binding of heterochromatin protein (HP1) and chromatin condensation
Mech of DNA methylation
Actin chromatin –> DNA methyltransferase –> MECP2 and sin3/Histone deacetylase recognizes methy. cytosine –> lysine methyltransferase –> HP1 –> condensed chromatin
Case Studies
- A1 methylated/silenced –> A2 expressed
- B1 silenced without enhancer binding
- -> ICR + enhancer –> A1 expressed - ICR methylated + no binding with enhancer –> enhancer binds B2 to express; A2 silenced
Hemizygoity
- Gene 1 (M exp) + Gene 2 (P exp) –> Fetal/adult somatic
- Germline phase (Methyl marker erased)
- Germline establishment (sperm and egg) is when methyl marker returns
- zygotic Fertfilization (Returns to 1)
Prader-Willis (1/12500, death by 30)
- M silenced, mutated P
- Reduced muscle tone, milk suckling, fertility and motor skills; increased weight gain
- Decrease in SNORD116 (Smaller nucleolar organizing RNA gene) 29 copies; SNORD115 –> autism
- Types
> 3-5Mb deleted in P 15q11-13
> Maternal disomy at Ch15
> small 15q11-13 deletion –> silencing
Angelman (1/16000)
- P silenced, mutated M
- Developmental delay, outburst of laughter, low mental capacity, happy
- Types:
> deleted UBE3A (ubiquitin protein ligase E3A) Ch15
> paternal disomy at Ch15
> UBE3A silencing (simple mutation/P-mutation)
X-Inactivation (All mammals, female selective)
- Late blastocyst of Xi –> calico
- XIST at xq13.2
> 17 kb non-coding, spliced and Poly-A RNA
> Hypermethylated Xi CpG
> Hypomethylated histones tight (lots of macroH2A1.2 histones) –> Xi DNA + Barr Bodies –> most genes on Xi silenced and replicated - Results:
> 1:1 of two cell types in females
> Skewing (75-95%) inactivation of same chromosome –> recurrent spontaneous abortion, anemia, extreme pigment defect, deafness
Certain X- genes are always expressed
- Long Interspersed Nuclear element (LINE1): XIST-specific recognition site (in rest of q arm)
- Genes that escaped XIST binding in Xp and/or Y (no dosage compensation)
Environmental Influence
- Twin genome: same DNA, different methylation
> Age-related: depends on time spent apart - Rats exposed to stress –> pathologically aggressive adults; Maternal care
Cell Lines
- Primary: not immortalized
- Established: immortalized; media + space in Nitrogen
> Transfection: cells take up DNA; plasmid DNA for GE –(endocytosis)–> host cytoplasm degradation
> Transduction: virus (target gene + promoter) in cell; microinjection of DNA (tedious)
GFP/Luciferase
- Tell tissue-specific promoter activity (cDNA)
- determines GE during development
- Fuses to protein –> chimeric –> study cell localization
In Vitro Organoid Culture
- Whole: bud-branching after dissection
- Miniature Tissue: mammary gland + lymph node + 3D
- Stem cell and 4. Primary cells + culture insert
Transgenic Mice (KO) *All DNA in GL cells (genetically modified)
Homologous Recombination
1-2-3-4 + NEO-DTA –> 1-2-NEO-4 + 3 (intron)
> DNA insert from HR into ICM of blastocyst from ES cell line –> select –> reimplant into foster mother
> Egg –> rotational cleavage –> 4-cell –> morula –> tight junction in compacted morula –> grow in mature blastocyst with 64-200 cell stage and ICM –> cross with heterozygote to identify AOI
Transgenic Mice (KI)
- UBE3A-YFP for Drug Screen:
> Topoisomerase reactivates UBE3A-YFP –> Pat-gene not silenced to make up for mutated Mat gene - Expressed exchanged gene at WT locus (HDR)
DNA Construct
- Strong DNA promoter activates cDNA transcription
- Microinjected DNA in fertilized eggs (pronuclear stage with nicked cDNA and DNA clone integration) –> transgene insert, cultured and implant in embryo
- Detect overexpression, find key regulatory element, and express particular protein
Immunodeficient Mice (No transplant rejection)
- NSG mice: no B cells, mature T cells and NKT cells
- PDX (patient-derived xenograft): tumor tissue grown in ID mice to mimic environment
- Humanized-xenograft modes: co-engraft tumor and peripheral BM/blood cells into NOD/SCID mice to reconstitute murine IS cells
CRISPR-Cas9 (Sequence-specific gene control)
- Spacer matches foreign sequence
- Cas9: DNA endonuclease guided by 2 RNAs
> protospacer: short foreign sequence fragment
> PAM: protospacer adjacent motif
> Now combines tracrRNA and crRNA
