6: Transgenics 1

Question 1

gene copy number: bacteria vs. humans?

Answer 1

bacteria: haploid, one copy of each gene. humans: dipoid, two compies of each gene

Question 2

dominant vs. recessive mendelian genetics?

Answer 2

dominant: only one copy in a diploid genome is altered. usually gain of function. recessive = both copies need to be altered. usually loss of function

Question 3

what do you need for pronuclear microinjection (5)?

Answer 3

construct (DNA for transgene). transgene specific probes to prove correct mRNA expression. transgene specific antibodies. mouse eggs. mouse foster mothers

Question 4

3 “DNA ingredients” in a transgene

Answer 4

promoter. ORF, open reading frame = protein coding region usually from cDNA clone. 3’ untranslated region and polyadenylation site.

Question 5

mode of DNA integration: 4 steps

Answer 5

microinjection of transgene construct. head to tail concatermerization (repeats). random chromosomal breakage. transgene insertion.

Question 6

how well do minigene transgenes work?

Answer 6

not well: increasing transgene copy number: no change in transgene expression level

Question 7

what type of DNA better for making transgenes?

Answer 7

big pieces of DNA. increasing transgene copy number = increasing expression level

Question 8

why is bigger transgenes better?

Answer 8

minigenes can end up in tighter packed heterochromatin. bigger transgenes can organize their own chromatin domain.

Question 9

4 attributes of a DNA construct to make a constitutive transgene

Answer 9

enhancer + LCR (locus control region). promoter. ORF = protein coding region. 3’ untranslated region/polyadenlation site

Question 10

transgene size: types of DNA

Answer 10

so you have your chromosomal gene: you can have YAC clones (yeast artificial chromosome). BAC clones (bacterial). cosmid clones (derived from bacteriophage). also minigene clones

Question 11

best type of transgene to use? (3 reasons)

Answer 11

cosmid clone: low to medium yield, high accuracy of gene expression and possible to produce high gene expression.

Question 12

sizes of YAC vs. BAC vs. cosmid vs. minigene

Answer 12

YAC = 300 kb. BAC = 150 kb. cosmid = 40 kb. minigene = 5 kb.

Question 13

how do you get mutations into construct? (2)

Answer 13

transgenesis = adding genes. homologous recombination = swapping genes.

Question 14

transgenesis involves?

Answer 14

isolate construct by DNA cloning from patient with mutation. site directed mutagenesis in plasmid clone with synthetic oligonucleotides. humologous recombination in e.coli or yeast

Question 15

how to get mutations from a plasmid directly into endogenous chromosomal gene?

Answer 15

knock in approach. homologous recombination.

Question 16

5 methods for phenotyping

Answer 16

transgene specific probes for northern blot, RNA protection assay. in situ hybridization. transgene specific antibodies. immunohistochem. neurological phenotypes

Question 17

conditional transgene: how?

Answer 17

Tetracycline-Controlled Transcriptional Activation. reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).

Question 18

how to make knock in of dominant mutation

Answer 18

floxed selectable marker gene

Question 19

recessives: usually what happens to protein

Answer 19

absence or reduced function of a protein.

Question 20

4 things needed to make knock out mice

Answer 20

mouse emybronic stem cells. mouse blastocytsts. pices of genomic DNA from gene of interest. selectable marker genes.

Question 21

selectable marker genes: use depends on?

Answer 21

the concept that genes linked together are inherited together

Question 22

selectabl e marker genes confer?

Answer 22

dominant resistance to a drug added to the cell culture medium.

Question 23

neo gene

Answer 23

neomycin phosphotransferase. neo gene attached with gene fragment of interest in one continuous piece of DNA. if cell takes up DNA it will survive because drug resistant.

Question 24

mutant gene/proteins involved in premature aging?

Answer 24

DNA repair enzymes and helicases. nuclear proteins called lamins. telomere shortening. CTC1 (works with DNAa prismase). calcium homeostasis: klotho)

Question 25

when you model a dominant mutation: transgenic? knock in?

Answer 25

transgenic kives higher levels of protein production. knock in gives low expression that exactly recaptiulates that of the parental locus

Question 26

how would you model a recessive mutation?

Answer 26

knock out

Question 27

klotho

Answer 27

secreted anti-aning factor