k********n
发帖数: 756 | 1 如果顾虑HETEROZYGOUS KNOCK-IN会导致整个动物因为神经功能问题不能繁殖，
HOMOZYGOUS肯定会死，整体设计思路应该是怎样？CONDITIONAL KNOCK-IN? | z****u
发帖数: 1007 | | S******9
发帖数: 2837 | 3 你这个想overexpression还是knockdown啊？
做flox老鼠吧 | k********n
发帖数: 756 | 4 需要做KNOCK-IN OVEREXPRESSION, KNOCKOUT 没有表型。 | H****N
发帖数: 997 | | w*e
发帖数: 740 | 6 overexpression does not have to use knockin at ROSA26 locus. Transgenic
approach can achieve higher dosage.
【在 k********n 的大作中提到】
: 需要做KNOCK-IN OVEREXPRESSION, KNOCKOUT 没有表型。
| l****y
发帖数: 486 | 7 Assume your knock-in here refers to mutating a site at the endogenous locus
(thus over expression-type approach, such as making transgenic mouse from
Rosa26 locus, will not be applied here).
Then the question is: you expect your mutation is a gain-of-function
mutation or loss-of-function mutation.
If your mutation is GOF mutation, it is easy, see Kras V12 KI strategy--
basically insert stopper element LSL before Kras V12 Exon. Note that the
allele LSL Kras V12 (before crossing with Cre) is a null allele, because
stopper will disrupt Kras expression, so you have to use heterozygous mice
in your study---After you cross with cre, You have 50% WT Kras, 50% Kras V12
. however
, this is fine because you are studying a GOF mutation.
If you are studying a LOF mutation (say, study a kinase dead mutant), then
the same conditional KI strategy mentioned above cannot be applied here.
first, you cannot use homozygous Conditional KI mice, because phenotypically
they are total KO. If you use heterozygous mice, then eventually the mice
have half WT kinase, half kinase dead mutant---you might not see any
phenotype(unless you expect strong dominant negative effect).
As far as I know, currently there is no good way to use conditional KI to
study LOF mutant.
【在 k********n 的大作中提到】
: 如果顾虑HETEROZYGOUS KNOCK-IN会导致整个动物因为神经功能问题不能繁殖，
: HOMOZYGOUS肯定会死，整体设计思路应该是怎样？CONDITIONAL KNOCK-IN?
| k********n
发帖数: 756 | 8 Thanks a lot, Lumphy.
The mutant has dominant-negative effects and we would like to make a
inducible KI (so it is GOF) since I am sure homo will die and hetero will no
be able to breed. We would like to have rat line instead mice due to its
larger size for microsurgery on infant animals and better characterized
behavior.
Mutating at the endogenous locus or over-expression at Rosa26 locus will, I
believe, give the same phenotype. Which one is easier, simpler and faster?
Regarding the Kras v12 strategy, would you give me a reference?
locus
V12
【在 l****y 的大作中提到】
: Assume your knock-in here refers to mutating a site at the endogenous locus
: (thus over expression-type approach, such as making transgenic mouse from
: Rosa26 locus, will not be applied here).
: Then the question is: you expect your mutation is a gain-of-function
: mutation or loss-of-function mutation.
: If your mutation is GOF mutation, it is easy, see Kras V12 KI strategy--
: basically insert stopper element LSL before Kras V12 Exon. Note that the
: allele LSL Kras V12 (before crossing with Cre) is a null allele, because
: stopper will disrupt Kras expression, so you have to use heterozygous mice
: in your study---After you cross with cre, You have 50% WT Kras, 50% Kras V12
| l****y
发帖数: 486 | 9 You can check Tyler Jacks, 2003 Cancer Cell paper.
Transgenic approach in general is easier, cheaper and faster, but may be
criticized for its intrinsic overexpression limitation. For example, Kras
mutant over expression and expression at endogenous level (by KI) lead to
different cellular phenotypes, but KI mimics more what happens in cancer
patients.
no
I
【在 k********n 的大作中提到】
: Thanks a lot, Lumphy.
: The mutant has dominant-negative effects and we would like to make a
: inducible KI (so it is GOF) since I am sure homo will die and hetero will no
: be able to breed. We would like to have rat line instead mice due to its
: larger size for microsurgery on infant animals and better characterized
: behavior.
: Mutating at the endogenous locus or over-expression at Rosa26 locus will, I
: believe, give the same phenotype. Which one is easier, simpler and faster?
: Regarding the Kras v12 strategy, would you give me a reference?
:
| x********e
发帖数: 35261 | 10 loxp STOP loxp gene应该是最保险的吧
【在 k********n 的大作中提到】
: 如果顾虑HETEROZYGOUS KNOCK-IN会导致整个动物因为神经功能问题不能繁殖，
: HOMOZYGOUS肯定会死，整体设计思路应该是怎样？CONDITIONAL KNOCK-IN?
| | | x********e
发帖数: 35261 | 11 LOF的情况下WT allele被floxed就可以了吧？
locus
V12
【在 l****y 的大作中提到】
: Assume your knock-in here refers to mutating a site at the endogenous locus
: (thus over expression-type approach, such as making transgenic mouse from
: Rosa26 locus, will not be applied here).
: Then the question is: you expect your mutation is a gain-of-function
: mutation or loss-of-function mutation.
: If your mutation is GOF mutation, it is easy, see Kras V12 KI strategy--
: basically insert stopper element LSL before Kras V12 Exon. Note that the
: allele LSL Kras V12 (before crossing with Cre) is a null allele, because
: stopper will disrupt Kras expression, so you have to use heterozygous mice
: in your study---After you cross with cre, You have 50% WT Kras, 50% Kras V12
| l****y
发帖数: 486 | 12 Not sure about your point here.
For example, if you are studying a kinase function in neurobiology. You
found total KO of your gene leads to embryonic lethal phenotype, and
conditional KO of your gene in neuron leads to very interesting phenotype.
Culturing of KO neuron in vitro also showed dramatic phenotype.
Interestingly, restoration of kinase dead (KD) mutant in KO neuron fully
rescued the phenotype (same as restoration of WT).
You wonder whether the neuronal phenotype you observed in vivo is kinase
function dependent or not, so you first generated a germline kinase dead
knock-in (KI) mice (in which you only mutated one residue in kinase domain
which is critical to maintain kinase function). However, KI/KI homozygous
mice is lethal (because kinase function is important for embryo development)
, while KI/+ heterozygous mouse are normal (because 50% WT kinase in the
cell can still do its job).
Now you want to generate a conditional KI mouse in which you can switch WT
protein to KD protein in adult neuron and see what happens. This is the same
idea for conditional KO approach: as long as you have an appropriate Cre,
you can switch off the expression of your gene at specific time and place.
However, as far as I know, there is no straight forward conditional KI
approach to afford this purpose.
The loxp-stop-loxp (LSL) approach cannot be applied here. Because LSL-KD
allele is a null allele. If you generate LSL-KD/+ mice, after you cross with
cre, you generate KD/+ genotype in neuron. You will not expect to see any
phenotype, because 50% WT kinase can still do its job (this is different
from LSL Kras KI/+ case, because Kras mutation is a gain-of-function
mutation, the mutation can have effect even there are 50% additional WT Kras
). If you generate LSL-KD/LSK-KD homozygous mice, it is lethal. You cannot
obtain such viable mice.
You can use transgenic approach to induce the expression of your kinase dead
mutant in neuron. However, this is not knock-in we are talking about here
and carries its own limitation as I mentioned above.
【在 x********e 的大作中提到】
: LOF的情况下WT allele被floxed就可以了吧？
:
: locus
: V12
| x********e
发帖数: 35261 | 13 Cre, LSL-KD/+没表型应该是fertile的吧？把这个line跟floxed WT line cross，得到
Cre, LSL-KD/floxed WT。如果用合适的Cre，能否得到KD/-？
【在 l****y 的大作中提到】
: Not sure about your point here.
: For example, if you are studying a kinase function in neurobiology. You
: found total KO of your gene leads to embryonic lethal phenotype, and
: conditional KO of your gene in neuron leads to very interesting phenotype.
: Culturing of KO neuron in vitro also showed dramatic phenotype.
: Interestingly, restoration of kinase dead (KD) mutant in KO neuron fully
: rescued the phenotype (same as restoration of WT).
: You wonder whether the neuronal phenotype you observed in vivo is kinase
: function dependent or not, so you first generated a germline kinase dead
: knock-in (KI) mice (in which you only mutated one residue in kinase domain
| l****y
发帖数: 486 | 14 Yes, this is the best we can do currently using LSL to study KI. I previousl
so basically, before crossing with cre, the target cells have 50% WT protein
, after crossing with cre, the target cells have 50% kinase dead protein.
Do you know whether there is any such publication using this mating strategy?
【在 x********e 的大作中提到】
: Cre, LSL-KD/+没表型应该是fertile的吧？把这个line跟floxed WT line cross，得到
: Cre, LSL-KD/floxed WT。如果用合适的Cre，能否得到KD/-？
| x********e
发帖数: 35261 | 15 我不是做老鼠的，不太清楚
previousl
protein
strategy?
【在 l****y 的大作中提到】
: Yes, this is the best we can do currently using LSL to study KI. I previousl
: so basically, before crossing with cre, the target cells have 50% WT protein
: , after crossing with cre, the target cells have 50% kinase dead protein.
: Do you know whether there is any such publication using this mating strategy?
|
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