Genetic Manipulation pt1

Question 1

pluripotency

Answer 1

ability of a cell to contribute to any tissue in the body

Question 2

pluripotent teratocarcinomas

Answer 2

“germ cell cancers”

tumours formed by germ cells (oocyte/sperm)

Question 3

embryonal carcinoma cells

Answer 3

EC cells
cells of testicular teratocarcinomas, can be kept in culture

e.g. F9 cells

Question 4

teratocarcinoma cells are pluripotent, proven by?

Answer 4

1. if take cells & transplant under skin of immunodeficient host mouse, they form tumour of various tissue types
2. if introduce F9 cells into mouse blastocyst in vitro and implant into pseudopregnant mouse they will give birth to chimeric offspring which have contributions from original blastocyst and EC cells

Question 5

ES cells can be derived from…

Answer 5

dissociating a blastocyst and culturing the ICM

Question 6

how do we know ES cells are pluripotent

Answer 6

can be made to differentiate into many cell types in culture

will form teratomas if introduced into adult mice

will contribute to all parts of embryo if reintroduced into blastocyst

Question 7

pluripotency genes

Answer 7

Oct4:
transcription factor expressed in ICM cells
Oct4 null embryos develop to blastocyst but then die as ICM not pluripotent

Nanog:
expressed in ICM cells
Nanog-null ICM cells lose pluripotency and develop extra-embryonic tissues (parietal ectoderm)

Question 8

how to maintain pluripotency of ES cells in culture

Answer 8

need leukaemia inhibitory factor (LIF):
signals through signalling pathways (JAK-STAT, Map kinase, PI3-kinase) to maintain Oct4 expression and prevent differentiation to mesoderm/endoderm

Bone morphogenic protein (BMP):
prevents differentiation into neuroectoderm

Question 9

what happens if withdraw LIF from ES cells on culture

Answer 9

embryoid bodies form - contain differentiating cells

Question 10

genetic manipulation of ES cells

Answer 10

ES cells can be genetically manipulated in vitro and then introduced into mouse blastocyst which can be implanted into pseudopregnant female mouse

have chimeric offspring which can then breed to produce fully genetically modified offspring

Question 11

electroporation

Answer 11

cells bathed in solution of new gene
electrocourette gives electric shock, blowing tiny lesions in cell membrane. they heal in milliseconds but gives time for new DNA to enter and get to nucleus

Question 12

reasons why genetic manipulation is done

Answer 12

understand basis of human, health & disease
identify the role of specific genes in disease
understand what drives been regulation
create designer animals for research, medical or other uses e.g. disease models

Question 13

examples of useful spontaneous mutations

Answer 13

Pax6 mutation: pax6 is a transcription factor, mice has small eyes, important for eye and brain development

Vangl2 mutation: important for neural tube closure, mice without fail to develop proper tails

Limk1 mutation: Limk1 is an axon guidance related enzyme. sciatic nerve doesn’t growth properly in this mutation

Question 14

random mutagenesis: how

Answer 14

exposing animals to a chemical mutagen

ENU (ethylnitrosourea):
creates point mutations by ethylating DNA pairs during DNA replication in sperm cells

EMS (ethyl methanesulphonate):
turns G/C pairs to A/T during DNA replication

Question 15

mutagenesis screens

Answer 15

mate mutated males with WT females

offspring heterozygous for dominant gene will show a phenotype

for recessive, mate offspring to create homozygous babies

Question 16

homologous recombination

Answer 16

usually occurs during meiosis

maternal and paternal copy of each chromosome line up and identical sequences of DNA find eachother and cross over

can happen anytime in cells when identical DNA sequences find eacoterh and line up

can be used to introduce new DNA into cells

Question 17

gene knockout by homologous recombination

Answer 17

flank new gene with DNA that surrounds current gene. The target vector should also have a identifier gene (neomycin resistance, GFP) and thymidine kinase gene outside the flanking sequences.

electroporate target vector into ES cells

add neomycin, cells that haven’t taken up the DNA will die

retainment of TK gene means the new DNA was randomly integrated but if TK is not taken up and remains on target vector then HR occurred –> five ganciclovir to kill cells with TK

do PCR

inject new ES cells into ICM of host blastocyst , implant that into female mouse –> chimeric offspring –> breed offspring to create homozygous knockouts

Question 18

transgenic animals

Answer 18

contain an exogenous gene introduced artificially

generally all cells contain the transgender and genetic change is heritable

Question 19

GM: can make animals that..

Answer 19

contain mutant forms of gene of interest
overexpress gene of interest, or express it in different tissues
express important products e.g. insulin in milk
express genetic markers e.g. GFP to study control of gene expression

Question 20

building a transgene - what a gene needs to work…

Answer 20

promoter sequence driving expression in appropriate tissue (transcription factors bind)
open reading frame encoding gene want expressed and bit encoding amino acids that line up to form protein
sequences that ensure correct mRNA processing e.g. polyadenylation signal so mRNA gets polyadenylated tail

Question 21

different methods to introduce trans genes into animal

Answer 21

direct injection DNA
chemical transfection
electroporation
viral infection

Question 22

chemical transfection of DNA

Answer 22

incubate cells in culture medium containing DNA and a chemical that wraps up the DNA
will either endocytose or diffuse through cell membrane

Question 23

how to increase chance that all cells of transgenic animal will carry the transgene

Answer 23

introduce transgene into the one cell embryo

(if inject into one cell of an 8 cell embryo, get a mosaic)

Question 24

direct injection of DNA

Answer 24

inject DNA into male pronucleus before it fuses with the female one

DNA repair mechanisms will recognise free DNA and hopefully integrate it into host DNA

Question 25

direct injection of DNA: integration of transgene into injection

Answer 25

integration random DNA may be chewed up by repair enzymes instead may put DNA into different gene

Question 26

direct injection of DNA: what if expression is not as expected

Answer 26

weak promoter/insufficient regulatory sequences copy number - don't know how many copies going in site of integration - DNA may be inserted near another gene and be controlled by that genes promoter epigenetic modification - cells think new DNA is viral & shut it down

Question 27

3 genes needed for virus to infect and make more virus s

Answer 27

gag - encodes proteins of nucleoprotein core pol - encodes reverse transcriptase and integrase Env - encodes surface protein components of virion packaging signal allows it to be wrapped up in new viral particle

Question 28

how to use a retrovirus to get transgene into animal cells

Answer 28

genetically engineer transgene to look like retrovirus - retroviral flanking sequences and packaging signal introduce this to helper cells, which contain genes encoding gag, pol and env, in vitro now have new viral particles made with transgene RNA --> can be used to infect cells

Question 29

cons of viral delivery

Answer 29

may only work in dividing cells cells spot virus and shut them down , longterm expression is a problem side effects (recombination leads to infective new viruses) safety considerations viruses are excellent way to introduce genes to cells in culture or to particular tissues of an animal in vivo not a good way of getting DNA integrated into whole animal (often silneced)