gene manipulations in organisms

Question 1

define transgenesis and how its delivered

Answer 1

controlled transfer of genetic information into the genome, with no reliance on selective breeding
often done by microinjection

Question 2

what is meant by forward genetics

Answer 2

phenotype driven
random mutation, phenotype change observed, gene identification, and then interpret gene function

Question 3

what is meant by reverse genetics

Answer 3

gene driven
gene identification, targeted mutation, phenotype change observed and then interpret gene function

Question 4

what is meant by intermediate genetics

Answer 4

gene trapping
random TAG insertion, gene identified and phenotype change observed, then gene function is interpreted

Question 5

why is a balancer chromosome often added in drosophila genetics?

Answer 5

it prevents crossover
contains identity markers
lethal recessive allele– thus no interference of natural selection

Question 6

what is the hedgehog mutation?

Answer 6

Hh mutation
in flies, mutation causes failure of separation of the cerebral hemispheres

Question 7

what is the one-eyed pinhead (Oep) mutation?

Answer 7

severe cyclopia in zebrafish, (one eye in the middle of the face)

Question 8

what is the Weissherbst (weh) mutation

Answer 8

hypochromic blood (less hemoglobin/red in RBC) and decreased blood cells – often in zebrafish

Question 9

what is the Hagoramo (hag) mutation

Answer 9

disrupted stripe pattern in zebrafish

Question 10

what is meant by synteny

Answer 10

same genes but in different order in the chromosomes accross species

Question 11

what are advantages of the mouse model (7)

Answer 11

• small
• reproductive efficiency (also non seasonal)
• tolerant to inbreeding
• small oestrus cycle ~ 4days
• 19 pairs of autosomes and X and Y
• all telocentric chromosomes
• same gnome size as humans – synteny

Question 12

what is ENU (N-ethyl N-nitrosourea)?

Answer 12

a supermutagen– high efficiency
transfer of ethyl group in DNA resulting in mispairing
induces single base pair substitutions

Question 13

what is a congenic strain, and explain what happens in gene linkage and non-linkage

Answer 13

when an inbred strain contains a small genetic region from another strain
linked– cause retention of small part of the other gene
no linkage– rapid elimination of heterozygosity

Question 14

what are two markers used to detect mutations in mice?

Answer 14

• microsatellite markers
• single nucleotide polymorphisms (SNPs)
  (often used to distinguish inbred mouse strains)

Question 15

how is gene mapping or linkage analysis done?

Answer 15

1) homozygous mutant(marker added) X homozygous wildtype
2) back-crossing x10
–> the location of the mutation from the residual chromosome segment left will be the CANDIDATE REGION
4) the mutation can then be identified by PCR

Question 16

Microinjection DNA is derived from what?

Answer 16

• plasmid
• bacterial artificial chromosome
• yeast artificial chromosome

Question 17

in transgenesis where is the egg from?

Answer 17

superovulating female mated to a stud male

Question 18

in transgenesis where is the male pronucleus from?

Answer 18

male pronucleus from a fertilized egg

Question 19

when does implantation occur into pseudopregnant foster female in transgenesis?

Answer 19

after blastocyst formation

Question 20

what influences does transgenesis have? (4)

Answer 20

• position effect– integrates at random location in ch
• mutagenic– may disrupt endogenous genes
• multicopy arrays– recombines to form concatemers before integration
• integrity– sometimes partially degraded before integration

Question 21

what is meant by transfection of ES cells

Answer 21

select for genetic change and reintroduce cells into embryo

Question 22

what is meant by chimera

Answer 22

combining 2 pre-implantation embryos and producing a single animal (eg 2 colours in coat)

Question 23

what are the three layers of the blastocyst and what to they develop into?

Answer 23

epiblast– foetus (internal cells)
hypoblast– yolk sac (layer covering epiblast)
trophoblast– placenta (layer surrounding all)

Question 24

what is the biggest origin of Embryonic Stem cells and what kind of potency do they have?

Answer 24

Epiblast (develops into foetus) and is pluripotent– often derived 3 days after fertilization

Question 25

what are feeder cells/fetal calf serum/ LIF

Answer 25

ES cells + non-dividing embryonic fibroblasts
– layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate

Question 26

what is isogenic DNA

Answer 26

identical cells– in transfection identical to ES cells

Question 27

what type of cell increases targeting frequency in the transfection of ES cells

Answer 27

isogenic cells, it increases exponentially

Question 28

outline the gene-targeting steps

Answer 28

1) transfect vector DNA– growth
2) selection of drug resistant colonies
3) growth of clones
4) plated on wells and run either
- southern gel blot
- PCR

Question 29

main differences between PCR and Southern Gel Blot

Answer 29

PCR
- primer complementarity to selection markers
- amplifies region
- PCR product is from target exon to the next
Southern Gel Blot
- restriction sites need to be present in target locus
- digests
- Southern gel blot product includes both homology arms

Question 30

name the three modern approaches to transgenesis

Answer 30

• zinc finger nucleases (ZFN)
• transcription activator-like effector nuclease (TALEN)
• cluster regularly interspersed short palindromic repeat (CrisPR)/ cas9 system

Question 31

how does CrisPR/Cas9 system work

Answer 31

1) cas9 endonuclease and guide RNA cleave DNA strands
2) gRNA contains the target site (20nt protospacer) and is upstream of the PAM site (NGG)
3) cas9 recognizes PAM and makes a ds break 3nt upstream from PAM
then can introduce genetic info in different ways

Question 32

what are the three different ways CrisPR/Cas9 system can introduce genetic info?

Answer 32

• NHEJ– non specific
• 2 gRNA target sites can be chosen– deletes large genomic sequence
• HDR (homology-directed recombination)
  use short oligos or DNA vectors with short homology arms that flank cleavage site– more specific and precise

Question 33

what is the more specific mutant of Cas 9 used to reduce off-target effects, how does it work?

Answer 33

mutant cas 9– ‘nickase’–cas9D10A
- cleaves only 1 strand
- uses two gRNA and 2 cas9D10A to generate overhand in both strands

Question 34

what kind of deletion can you use to determine specific mutation in specific stage in development?
what molecule is released?

Answer 34

Inducible mutation
- cre-ERTZ + Tamoxifen added and moves into the nucleus causing recombination
HsP9O often released from cre-ERTZ when this occurs

Question 35

why are maternal and paternal pronucleus required for normal development?

Answer 35

due to IMPRINTING and monoallelic uniparental expression
some genes are expressed/repressed depending on parental origin, often complementarily expressed

Question 36

what are the two types of somatic cell nuclear transfer (cloning)

Answer 36

• reproductive cloning
• therapeutic cloning (ES cells cloned from embryo generating cells types/tissues)

Question 37

why is there such a low survival in cloning?

Answer 37

due to abnormal epigenetic regulation
- only partial reprogramming occurs, DNA methylation, chromatic remodeling, and zygomatic gene activation are all not fully completed