Same KO Mice by Different Techs (ES vs CRISPR) Show Different Phenotype... Why?

I would like to assess the editing efficiency of designed guide RNAs by transfection prior to establishing a stable KO cell line (via lentiviral infection). HEK293T cells are very efficient when assessing gRNA targeting human genes. However, I will be using a mouse model in parallel with the stable cell lines and would like a stable mouse cell line. Any suggested cell lines would be appreciated, along with the transfection media used and observed transfection efficiency.

In terms of the implications for using CRISPR/Cas9 to generate a knockout cell line, what are the major differences in doing so in HEK293 cells vs. generating the same knockout in HT-22 mouse hippocampal cells?

I am in the process of knocking out a gene in my U87 (glioblastoma) cells using CRISPR. I've been following the Santa Cruz protocol, however we are having a really hard time optimizing the protocol at the transfection phase. The moment we increase/decrease transfection reagent to match what we think is the best plasmid concentration, we either too much toxicity or very few green cells (gfp sensitive). We've tried a range of about 10% - 30% plasmid to reagent conc. Has anyone done this before? Any suggestions will be very much appreciated.

I am using Lentivirus CRISPR techniques to cut interested gene in primary cells. But I have a problem with virus titer. I would like to increase the titer to get high transduction efficiency. Is anyone can suggest me good lentivirus plasmid backbone that suitable for a CRISPR? 

Performed plasmid mediated CRISPR/Cas9 knockout of mir-208a in human cardiomyocytes. Sequencing suggests a heterozygous knockout. However, qPCR still shows equal expression of 208a within control and knockout cells. Negative control is clean, but amplification curve does not begin until cycle ~30. Will a mono-allelic knockout not reduce expression of mature microRNA?

Protein X is a synaptic protein.

CRISPR was perform and a frameshift with an insertion was obtained that resulted in a null KO and HZ KO. Protein was quantified using western blot to check in the null KO and no protein was found. Since it was the same mutation, it was assumed that the heterozygous KO also worked. Sequencing and quality contro were fine.

We performed electrophysiology and got no differences between WT and HZ. Moreover, synaptic staining showed that X was the same in HZ and WT. However, the KO had no protein X.

Now we are looking at the western blot, and it seems that HZ and WT have similar protein levels (not confirmed yet as it hasn't been quanitfied)

A previous paper reported 30% reduction in HZ condition as compared to wild type.

Is it possible that dose compensation is occurring here? it's non X chromosome gene. What are the other possibilites?

*We are also re-sequencing the lines now.

Hi all, 

I am interested in KO/KD of 2 genes, at first I want to test the effect of each

separately  (in a mouse cell line), but also I want to test the effect of a dual KO/KD. 

I have a good experience with shRNA, but I guess KO is always better than knock-down, specially when you want to learn about the genes rule. 

However after doing some reading, I understand that when working with the crispr system, one should test the results on difference clones, and from my experience, 

even without any manipulation almost each clone acts differentially  in many terms, hence the KO will be probably masked. Of course this can be overcome by testing few clones with KO (not always possible), but since today reviewers ask also to see the KO effect with different vectors) and I am interested in 2 genes separately  and also together, it will create plenty of groups, and this may be a problem, especially when I want to test the KO effect by implentinig the KO cells into mice. 

Please, help me get the right choice, is there any reason not to go with shRNA?

Thanks, Ran

Is there any protocol of knocking out one gene in THP1 cells using CRISPR/Cas9 system ?

Here, I used LentiCRISPR-v2 system to harvest virus carring guide RNA, but when I added the virus into THP1 cells, these cells were going to be activated and differenatiated, and they would grow together.

Also, THP1 cells are a little hard to be transfected with lipo2000, so pX459 or pX458 plasmids system may be not availiable.