a*******g
发帖数: 162 | 1 想GENERATE HOMOZYGOTE的TG MICE， 请问怎么可以GENOTYPING啊，用QPCR可靠吗？需
要用TAQMAN还是普通QPCR就可以了？多谢！ |
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m******5
发帖数: 1383 | 2 大家一般都筛多久呢？
筛出来有多少是干净的homozygote?
我用6mg G418 筛了25天就6，7个克隆…… |
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w********g
发帖数: 107 | 3 as your calculation:
( 9/50) x (1/2) x( 1/10) x (1/2)= 0.0045
but the correct answer in this question is (B) 0.045 =( 9/50) x (1/2) x( 1/
10) x (1/2)
Actually, 0.045 is the best answer here but not the perfect one.
I saw another version of this question as below:
A man is a known heterozygous carrier of a mutation that causes
hemochromatosis (AR). Suppose that 1% of the general population consists of
homozygotes for this mutation. If the man mates with somebody from the
general population, wh... 阅读全帖 |
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M*****n
发帖数: 16729 | 4 Basically, inbreeding dramatically increases the frequency of homozygosity,
which is the cause of lots of recessive genetic disorders in human being.
eg. regarding allele a, the ratio of homozygotes (aa) among the progenies of a
random mating is only a*a, while from an inbreeding you mentioned is 1/64.
Usually the recesssively pathogenic alleles are rare, and thus a<<1/8.
Therefore the risk for your gf to get two a alleles, which results in a
recessive homozygote, is much higher than the others. |
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S******9
发帖数: 2837 | 5 A man is known hetrozygous carrier of mutation that causes
hemochromatosis.(autosomal recessive disease).Suppose that 1% of general
population consist of homozygote for this mutation.If the man mates with
sombody from general population what will be the probability that he and
his mate will produce a child who is an affected homozygote.
a.0.025
b.0.045
c.0.09
d.0.10
e.0.25
B
q=1/10
p=1-1/10=9/10
female: 2pq=9/50
female transfer this gene to kids: 9/50x1/2
male transfer this gene to kids: 1/10x1/... 阅读全帖 |
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w*****n
发帖数: 107 | 6 我遇到过和你类似的情况，不过是我找别人要老鼠。对方给了，但是要authorship, 我
觉得reasonable.
"thanks very much for your note. We would be happy to send you the mice,
which have been thoroughly backcrossed on a C57Bl6 background. As you may
remember from the paper, they get pretty sick and don't breed as homozygotes
. An alternative might be the floxed allele, which we also have, and you
are welcome to. In general, we ask to be offered co-authorship on papers
using the mice, which I hope is not too onerous." |
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m****r
发帖数: 383 | 7 二倍体生物里面,通常每个基因有两个copy,也叫两个allele, 每个allele都可以自行生
产蛋白.两个allele相同的时候,这个二倍体生物叫homozygotes,不同的时候(通常一个
allele正常,另一个发生了基因突变),叫heterozygotes. loss of heterozygosity发生
时,正常的allele被丢掉,另一个带有突变的allele生产突变蛋白产物.这个产物可能仅
仅是缺失了正常功能(大多数情况),也可能在缺失正常功能的同时,获得一些新的功能,
遗传学上叫gain of function. |
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O******e
发帖数: 4845 | 8 I wouldn't count on that. lz doesn't really have to get the homozygotes. |
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D*a
发帖数: 6830 | 9 就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
with
to |
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D*a
发帖数: 6830 | 10 cre不是我们自己做的啊
我倒是知道有人鉴定cre homozygote是靠交配来鉴定的。
按这种思路，如果不牵扯background，倒是可以纯合杂和的cre一起用来交配下一代，
至少可以把下一代的cre+ 提高到 > 50% |
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i*********0
发帖数: 915 | 11 亲代tek-cre 又不一定是homozygote，子代可以是＋也可以是－。 |
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S*****s
发帖数: 287 | 14 这个难度比较高。你知道 transgene 插入在基因组的位置么？如果知道的话设计
Primer 扩充相应位置就行了。好奇的问一句，你为什么要 homozygous tg mice？ |
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R****n
发帖数: 708 | 15 syber green原理是用allele specific PCR吧，一个primer 3'端用来区分phenotype.
我没做过不太清楚效果。还有什么和ligation结合的，HRM的。你说的太笼统了。
Taqman是用probe,两边一个dye一个quencher，不同的sequence dye不一样，一管PCR搞
定。这个基本上是业界的标准方法 |
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c******r
发帖数: 3778 | 17 sybr green也可以，但是需要分开跑。
sybr green是个non-specific的DNA double strain dye.就是说，只要是PCR产物都可
以stain。所以没法用multiplex reation做internal control。
如果用taqman，pcr效率不错的话，可以设计两对primers，一对测你的target
sequence，一对做个其他随便什么基因的sequence。分别用不同的颜色标记。这样在一
个样品里跑两个pcr。如果如果target sequence是control的50%（一般给+-20%的误差
）就是single copy。如果和control一样（也是20%误差）就是two copies。
如果你的primers设计比较好，两对的amplification efficiency非常接近，那么可以
直接用ct数来计算二者的关系。但是如果两对primers的效率相差比较多，那么只能每
次都分别给两个primer做standard curve。这样保证二者的定量可比，也是可以的。就
是麻烦点。 |
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H***3
发帖数: 61 | 18 我的情况跟前一段时间讨论的一个情况不同。
转基因植物携带overexpression promoter+gene+c terminal GFP，转到野生型里建立
过量表达突变体。基因是核编码的叶绿体蛋白，蛋白N末端一个小酞段引导蛋白
targeting到叶绿体内行使功能。根据抗性筛选到homozygotes,confocal下检测到GFP信
号在叶绿体里，非常阳性。DNA genotyping和RT-PCR检测到基因插入正常，mRNA正常，
现在的问题是western用anti－GFP抗体无法检测到全长蛋白，只有GFP大概27kD左右的
信号。因为是未知蛋白，没有特异性抗体，突变体性状也没有发表过，所以无法确定是
否融合蛋白正确表达。我唯一能想到的是蛋白合成后，送到叶绿体内被降解了。可是即
使有降解，不会全都降解吧。
请大家给点意见，好不容易建立的突变体，实在不想放弃。 |
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y**********n
发帖数: 478 | 19 经典遗传学的东西怎么跟现代基因学联系起来有时候确实很头大。我试着解释下，先贴
一段wiki上的：
“An allele (UK play /ˈæliːl/ or US /əˈliːl/)
is one of two or more forms of a gene or a genetic locus (generally a group
of genes).[1][2] The form "allel" is also used, an abbreviation of
allelomorph. Sometimes, different alleles can result in different observable
phenotypic traits, such as different pigmentation. However, many variations
at the genetic level result in little or no observable variation.
Most multicell... 阅读全帖 |
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i*********0
发帖数: 915 | 20 6mg, this is really high?
which cell type are you using? |
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m******5
发帖数: 1383 | 21 mES cell
by the way, you still didn't answer the last question regarding whether or
not your mES cell could go into germ line………… I am really curious about
it. |
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i*********0
发帖数: 915 | 22 we did not try to get a germline transmission. |
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y******8
发帖数: 1764 | 23 You may get undesired integration. |
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g*********5
发帖数: 2533 | 24 usually how long could you maintaining the mES cells?
about |
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m******5
发帖数: 1383 | 25 could you make your question more specific? I don't get it…… |
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m******5
发帖数: 1383 | 26 Dear All:
I got a mice by hand with specific genetic trait, I want to get a homozygote
mES cell line from it. Knowing that we could not isolate mES cell from
blastocyst in our facility, I tunred to iPS or nuclear transfer.
IS there known facility out there provide service that we could make iPS or
nuclear transfered ES cell in a reasonable price range? thanks!!! |
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D*a
发帖数: 6830 | 27 晕你测heterozygote啊，不能先交一下交出homozygote来么，也就三个星期。
这个就是最大的问题了，hetero的两个带本来就序列一样，杂产物更多，而且又差不多
长，在胶上互相混尤其明显，你读到一半读不下去就是到了野生型位点了。最好是拿到
纯合老鼠测，实在不行你的野生带能不能酶切了？我们当时是等到纯合老鼠，酶切是我
自己想觉得行。不过怎么都要做nested pcr 才能测序。
wild |
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m******5
发帖数: 1383 | 28 插个题外的话，个人觉得Marcm的局限性比较强
但也许是理解有限。 比如如果这个homozygote的allelle影响globle cell migration
behavior 就无法用Marcm进行什么有价值的分析 |
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n**e
发帖数: 2026 | 29 同意，这是权威解释。前一段在这里；
The major hurdle that must be overcome in the development of new inbred
strains from wild populations is inbreeding depression which occurs most
strongly between the F2 and F8 generations. The cause of this depression is
the load of deleterious recessive alleles that are present in the genomes of
wild mice as well as all other animal species. These deleterious alleles
are constantly generated at a low rate by spontaneous mutation but their
number is normally held in check by th... 阅读全帖 |
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B*****l
发帖数: 261 | 30 测双亲看看有没有遗传病携带基因（隐性），然后看胎儿是homozygote or
heterozygote |
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C****n
发帖数: 79 | 31 Great,你说得到一个homozygote。 我之前也问相同的问题。这里再请教一下，那据你
分析，为什么会有相同的突变出现？ |
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T*G
发帖数: 600 | 32 同性恋本来就是遗传型的，好多发的小paper都是claim 同性恋在homozygotic twin 上
有遗传性，现在这么多lg, 其实做个gWAS一下就看出来了，现实是别说欧美了，中国有
人敢做吗。当然lg后天影响肯定也有室友的。 很多fitness 接近zero 的病一样从古到
今都有，为什么呢， fragile gene/hot spot/de nova 贝 |
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n***h
发帖数: 364 | 33 SUMO2009 and daisyy:
Do you mean the female is a hetrozygous carrier , not homozygote? |
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