Transgenesis or Germline Modification of Animals

Question 1

Germline Modification How To

Answer 1

Injection of DNA into 1 cell embryo
Stem cell (embryonic and IPS) modification
Cloning
DNA editing

Question 2

Transgenesis

Answer 2

aka genetically modified animal; genome has been experimentally altered by stable introduction of exogenous DNA or modified endogenous DNA in cells that form germline; will transfer altered genome to offspring -> permantely modified line of animals

Question 3

gene targeted

Answer 3

genomic modifications achieved by homologously recombination in embryonic stem cells

Question 4

transgenic animals

Answer 4

modificiations made through random and stable insertion of exogenous DNA into genome

Question 5

genetherapy

Answer 5

genetic modification of cells not in germ line (do not pass these on)

Question 6

why do we do germ line modificiations

Answer 6

• determine fx of individual genes
• produce models of naturally occurring dx
• produce proteins of medical or commercial value
• improve health and productivity of domestic animals
• examine cellular processes by introduction of signaling molecules
• species preservation (species at risk or threatened or preserve phenotypes ppl find valuable)

Question 7

why know about germ line modifications

Answer 7

• may treat transgenic animals in carer
• key element in stem cell therapy
• need ppl for animal sx, care, evalulation
• interest of clients
• focus of biomedical research, power to facilitate understanding of genetic and acquired dxs of animals
• Ethical issues

Question 8

Germ cells

Answer 8

egg sperm and precursors; haploid cells can combine and give rise to other cells

Question 9

somatic cells

Answer 9

non germ cells; genetic modifications to the cells not transfer to subsequent generations

Question 10

embryonic stem cells

Answer 10

derived from undifferentiated inner cell mass of early embryo at blastocyst stage; these are pluripotent (can generate many types differentiated cells); can be maintained in culture and retain pluripotency

Question 11

induced pluripotent stem cells

Answer 11

somatic cells that have been made pluripotent by introduction or activation of specific genes

Question 12

chimeras

Answer 12

animal formed from cells from different genetic lineages

Question 13

tissue specific stem cells

Answer 13

pool of undifferentiated cells that continually regenerate the organ- specific differentiated cells; have limits to n ability to generate other cell types (can’t become germ cells)

Question 14

single nucleated cell

Answer 14

modify with nuclear injection and CRISPR

Question 15

blastocyst

Answer 15

this is point of entry for embryonic stem cells

Question 16

Transgenesis by pronuclear injection of DNA

Answer 16

tandemy arranged copies of gene are inserted randomly into one chromosome in each cell; to direct expression of DNA need promotor, trans gene cDNA and a stop codon; inject into nucleus and this will enter DNA randomly; this is not most controlled way to make transgenomoic animals, can have mouse over or under produce it or interrupt important gene or silence it or get lots o expression; this is most widely applicable method (can be done I many species)

Question 17

elements of a transgene

Answer 17

1. cDNA not genomic DNA
2. Promotor considerations (determines pattern, timing, and extent of gene expression), enhancers increase degree of gene expression, sequences far removed form gene may play critical regulatory role
3. Polyadenylation signal (stop codon)

Question 18

Other method to produce genetic alterations by direct injection

Answer 18

1. BAC transgenesis (can inject engineerd bacterial artificial chromosomes fragments of genomic DNA, replace endogenous promotor)
2. Gene knockdown. Can inject RNAi or Dominant Negative transgenes

Question 19

summary of trans genesis by pronuclear injection

Answer 19

1. Mate mice at night
2. Harvest embryos in AM
3. Culture briefly and inject transgenic DNA into pronucleus of embryos
4. Culture overnight and transfer into psuedopregnant mouse in Am
5. Genotype offspring

Question 20

Features of trans genesis by nuclear injection

Answer 20

• multiple copies in head tail arrays; random integration
• rearrangements and deletions may occur
• random insertion may interrupt endogenous gene
• usually used to examine over expression phenotype or lineage tracing
• good for determining promotor structure/ activity
• can be used for loss of fx phenotype by dominant neg expression, siRNA expression, or insertional mutation
• Cell/ Tissue specific promotors confer selective gene expression

Question 21

Gene targeting by homologous recombination in ES (and iPS) cells

Answer 21

• can propagate ES cells and inject back into diff blastocyst and make chimeric animals
• these cells retain pluripotent but we’ve only been able to do this in mouse
• would have to put iPS in human embryo to prove as good as ES and we can’t do that

Question 22

how does gene targeting by homologous recombination work

Answer 22

take ES out and inject it; pick clones grow them and inject into blastocyt; these participate in informing cells of embryo including germ cells; need to get DNA to germ cell and know we are precisely modifying gene at location we want then need to confirm modification and clone it

Question 23

steps gene targeting by homologs recombination in ES (and iPS) cells

Answer 23

1. Make construct (w/ homology to region we want to modify)
2. Transfer into ES for homologous recombination, expand and select
3. inject into blastocyst
4. transfer to pseudopregnant female
5. Breed chimeras
6. Breed F1 heterozygotes
   7 Homozygotes

(if chimera extensive enough will lead to F1 heterozygote and then you breed F1 heterozygotes together to get F2 homozygotes then have permanately altered DNA line)

Question 24

Gene targeting in ES cells requires

Answer 24

line of embryonic stem cells that can be propagated culture w/o loosing ability to differentiate into germ cells

Question 25

for gene targeting in ES cells want to replace DNA in genome

Answer 25

at precise locus w/ DNA construct made in lab (allows specific mutations or deletions in ES cell allele)

Question 26

Replacement of DNA in genome of ES cell

Answer 26

involves delivering constructed DNA to cell and selecting for the v low probability homolgous recombination events where delivered DNA replaces endogenous DNA

Question 27

Gene targeting

Answer 27

can be gene inactivation or gene alteration; involves constructing target vector that contains DNA that is homologous to locus to be altered; currently only possible in mice ex. with simple codon modification can -> mouse having thing analogous to CF

Question 28

gene inactivation

Answer 28

- usually antibiotic restitance gene and gene encoding for protein that creates lethal metabolite of drug are included in the vector; will be expressed if stably inserted into ES cell genome - usually DNA vector will randomly insert into ES cell genome and in about 1% of cells hlogous DNA sections will bind to and replace endogenous DNA in process catalyzed by recombination enzymes which = pt of normal process resolving DNA breaks and mismatches cells will survive bc willl loose HSVtk (lethal metabolite gene) bc not between homolgous arms) and these cells will survive neomycin and will be selected in culture from non homologously recombined ES cells that die (gene is inactivated bc exon 3 is no longer present); check for gene in desired location by southern blotting

Question 29

Gene alteration

Answer 29

positive selection cassette placed outside exon flanked by recombination sites; desired mutation in exon 3 would be reproduced in targeting cassette; homologous recombination would be performed and ES cell clones selected and confirmed; recombined ES cells are injected into developing embryo at blastocyst stage then embryo -> psuedopregnant females and chimeric pups and backcrossed with normal mice and if alteration in germ line it will be in som offspring

Question 30

Features trans genesis by gene targeting in ES cells

Answer 30

- can selectively eliminate a gene - targeted placement of subtle mutations - injections easier but entire process is more labor intensive - can replace a gene w marker to ID timing and location of expression in vivo (ex add GFP) - can conditionally remove a gene in specific tissue or at specific time - adaptation during development can complicate interpretation

Question 31

nuclear transfer

Answer 31

= cloning; involves transfer of diploid nucleus from a cell to an enucleated unfertilized egg; have to then convince egg with genetics form somatic cell nucleus to thinking its single celled embryo by chaining conditions in cell so it thinks its single cell let it start dividing then put embryo into animal; way to produce targeted genetic modifications in mammalian species other than mouse (virtually all mamammalin species)

Question 32

limitations of cloning

Answer 32

gene expression is also controlled by epigentic factors and mitochondrial DNA which comes from host egg and these must be considered these may result in differences in phenotype btwn clone and nuclear donor and can -> abnormalities in cloned animal; cloning is v inefficient and most don't work

Question 33

Gene editing

Answer 33

- use CRISPR/ CAS9; repeat sequences guide nucleases encoded by CAS genes; CAS protein locates CRISPR repeat sequence which binds to homologous DNA; efficient gene editing system can be created w sequences homolgous to area of DNA to be edited linked to CRISPR repeated; binding of RNA to region of interest guides the CAS9 to DNA leading to 2x stranded cut at precise location; donor DNA added -> efficient gene editing can inactivate gene, correct a sequence, or add a new gene

Question 34

CAS9

Answer 34

nuclease that cuts DNA (put CAS9 recognition RNA we put it will help it bind homolgous RNA)

Question 35

Male germline modification

Answer 35

efforts being made to use spermatogonial stem cells (sperm stem cells) for trans genesis; haven't been able to maintain pluripotent

Question 36

somatic cell modification

Answer 36

- somatic cell gene therapy - antisense and RNAi methods - target somatic cells via viral infection, liposuction, DNA injection; gene expression often transient, may occur as non integrated episome or may stabile integrate

Question 37

ethics

Answer 37

- appropriate and acceptable rationales for reproducing species - limits to germline manupulations (Human germline?) - limits on production of transgenic animals (glow fish) - release and contamination of wild or domestic populations? - societal acceptance - patent transgenic animals allowed or not?

Question 38

features of trangenesis by nuclear injection into fertilized eggs

Answer 38

- random integration of multiple copies - promoter determines timing, extent, and pattern of expression - most commonly used method to produce animals that make drugs

Question 39

Features of transgenesis by homologous recombination in ES cells

Answer 39

- targeted genetic alterations - ability to produce precisely desired mutations - mouse only mammal we can do this with - conditional gene activation or inactivation is possible

Question 40

methods of transgenesis

Answer 40

- Nuclear injection into fertilized eggs - Homolgous recombination in ES cells - Nuclear cloning - Male germ line - Somatic cell gene therapy