FAQs - Frequently Asked Questions

HAPLOBANK Cell

  • How do we handle haploid ES cell lines?

    Basically as you would handle any other (ES) cell line, standard cell culture practice/techniques apply. Please check out the protocol section for general guidelines as to how the cells should be handled. For a general description, please see following publication: Elling et al. (2011). Cell Stem Cell, 9(6): 563-74. If you are interested, we offer ES cell culture training in our stem cell facility.

  • Which serum do you use?

    We usually perform batch testing of our serum to ensure continuous rapid proliferation and a high rate of colony formation of our ES cell lines without inducing differentiation. We usually test around 5-6 different batches. We recommend you either perform similar testing on any ES cell line which you may have available or alternatively use a serum that has been validated by the stem cell facility at your institute.

  • What should I use as control cell line in my experiments?

    The best control cell line to use would be a non-disruptive sister clone (genetrap positioned in antisense relative to the gene) which can be ordered together with the original clone. Alternatively, our wildtype cell line “AN3-12” can serve as negative control, which can be ordered (cell-ID is 00000WA00).

  • In which format do you supply the parental haploid ES cell line AN3-12 and clones from your collection?

    While our HAPLOBANK clones are frozen down into 2mL cryo vials, our parental wild-type cell line AN3-12 is contained in 2D Matrix tubes. Both formats contain enough cells for direct seeding onto a 10cm cell culture dish.

  • Do the cells stably maintain their haploid phenotype?

    No. Our haploid ES cells become diploid over time. It was estimated that ~2%-3% of haploid cells become diploid for each day in culture. Thus, they need to be regularly FACS sorted for their 1n status.

  • Does diploidization influence the knockout efficiency of targeted genes?

    No, as clones were mutagenized at their haploid state for which we select for by FACS before and after mutagenesis. Thus, diploidization is not a problem because the insertions will be present in both alleles. However, there is a chance that we mutagenized a diploid cell and it escaped our selection against diploidy. In this case, the cell line was not homozygously targeted and is not a knockout. This can be verified by performing the Integration PCR (see Integration PCR-protocol in the protocol section).

  • What tests should I run after receiving my clone-of-interest?

    After successfully reviving, expanding, and preserving the clone at your lab, we recommend to do some quality controls yourself. Firstly, you might want to start with the Barcode PCR to confirm that you in fact received the correct clone, which you do by simply amplifying the internal barcode of the clone at hand and comparing it to our database. Secondly, you can do an Inverse PCR to retrieve the integration site(s), and/or you can do the Integration PCR. The gene trap internal primers in combination with the clone-specific primers will only give you a PCR product if targeted correctly. Please, browse our protocol section for the protocols and more details on the different PCRs.

  • What quality controls were done before you shipped the clones?

    Every clone expanded, we routinely test for Mycoplasma-contamination using a qPCR assay. Only Mycoplasma-free clones get shipped.

    We also perform a Barcode PCR and an Integration PCR to confirm the correct identity and the correct genomic integration site of the clones.

    We provide you with a summary sheet of all quality control results when we ship the clones.

  • Where is the genetrap located relative to the "mapped sequence"?

    That depends on the mutagen: in Retro and Lentiviral clones the genetrap lies downstream of the mapped sequence, in Tol2 clones upstream.

  • What does it mean if Sanger Sequencing of the Barcode PCR product gives more than one Barcode for a clone?

    You will detect more than one Barcode for a clone if it carries more than one genetrap (i.e. grey clones). Compare the results to the Integration PCR and keep this in mind when working with this clone.

  • What do I do if the Integration PCR is not working and how do I interpret deviations from the expected pattern?

    First make sure that you chose the right combination of primers and DNA (see Integration PCR protocol). If you get no PCR product at all (not even in the cyan lane) consider using another polymerase, doing a new genomic DNA prep or designing new primers.

    If you only get a PCR product in the cyan lane compare the results to the Barcode PCR and consider performing an independent Inverse PCR.

    If you get the expected PCR products (yellow, cyan and red lane) and an additional product in the orange lane, showing the same size as the product in the cyan lane, the clone can be considered as heterozygous and is thus no true knockout. You should compare the results to the Barcode PCR and consider ordering another clone if available.

    If you get PCR products only in the orange and cyan lane (same size) the integration site is not confirmed. You should do an Inverse PCR and also compare the results to the Barcode PCR.

  • How do I best confirm that a clone is a true knockout for my gene of interest (GOI)?

    We suggest that you do two things: Firstly, you quantify the mRNA of wildtype and mutagenized clones for your GOI by qPCR. Comparing both samples, you should see a massive drop in expression for the latter, respectively. Secondly, you analyze your wildtype and mutagenized clones for your GOI on protein level and perform a Western blot. In case of a true knockout, complete loss of protein should be apparent in your knockout sample As for qPCR, you might want to design your primers in exons that flank the mutagen, e.g. if your clone carries the gene trap in the first intron, you would design your qPCR primers in Exon1 (fwd) and Exon2 (rev), if possible. Should you not be able to confirm the knockout of your GOI by both methods, please get in touch with us at office@haplobank.at to discuss alternative options.

  • What do the annotations in the column "Mutation" mean and what kind of mutation should I choose?

    The column “Mutation” provides information about the site of mutagen integration relative to the gene body:








    Due to several transcripts per gene an insertion can have several of these features. If several feature types are assigned to an insertion following priorities are recommended to be applied (S = sense, AS = antisense):
    1. 1Intron_S (if ordered together with Inverted Sister Clone _AS as well)
    2. Intron_S (if ordered together with Inverted Sister Clone _AS as well)
    3. CDS_S
    4. CDS_AS
    5. 5UTR_S
    6. 5UTR_AS
    7. ncExon_S
    8. ncExon_AS
    9. 3UTR_S
    10. 3UTR_AS
    11. Upstream_S
    12. Upstream_AS
    13. InterUp_S
    14. InterUp_AS
    15. InterDown_S
    16. InterDown_AS

    We regard a mutation as disruptive, if the mutagen is inserted in 5UTR, CDS, ncExon and 1Intron and Intron if inserted in sense. In 1Intron and Intron antisense clones the mutation can be easily converted to disruptive; to obtain these just click “Together with Inverted Sister Clone” when ordering. We would recommend going for an insertion in the first Intron (1Intron) if possible. Clones that carry the gene trap in an intergenic region (i.e. annotated as Upstream, InterDown, InterUp), cannot be regarded as knockouts of the respective gene. Please also keep in mind that a mutation in the coding sequence is phenotypically not reversible.

Shipment

  • How long does it take to get the ES cells clones?

    It usually takes us 4-6 weeks to expand the clones and do the quality controls. You will get an e-Mail notification when your clones are ready for shipment. Once the MTA and all clones are ready (clones are expanded and QCs are done) we will get in touch with you to ensure that you will be there to receive them the week after. Cells get shipped on Mondays, regardless of the destination. After we shipped the clones we will send you the tracking number by e-mail. In Europe the package is delivered within 2 working days. Shipments outside of Europe usually take a few days longer e.g. Japan 2-3 days, USA/Australia up to one week.

  • I would like to order ES cell lines. What are the shipping costs by express courier?

    We use a transport box which accommodates a plastic bag with clones and fill it up with 5-6 kg of dry ice, i.e. we use the same pack and dry ice amount for any cell line shipment regardless of the cell line quantity. The estimated FedEx shipment fees to the USA would be around € 270, and around € 150 for destinations in Europe. Please contact your local delivery service for detailed information about shipping costs.

  • Do I need an import permit to get the ES cells?

    Please inquire with your local authorities and make sure that we have all required documents in place for successfully shipping the cells to your country (i.e. TSCA for US import). This may also require special courier services as e.g. FedEx may not ship a dry ice parcel to any country (i.e. China). Should your country require special permissions to import murine ES cell lines (or any cell line for that matter), please make sure that you have all required documents in place. The same holds true for shipping labels, should they be required. HAPLOBANK will not replace cell lines, which have been refused at the customs because of missing or expired importing documents, and will likewise not cover any costs associated with such a delay in delivery.

  • Can the shipping address be different from the address on the import permit labels?

    It is not recommended that you borrow and provide us with the shipping information from a colleague, because this might cause confusion at the customs (or import regulation agency) and result in a massive delivery delay. In this case, HAPLOBANK will not replace any cell lines which did not survive the shipment.

  • Why do you use only DHL/FedEx and not postal service?

    We have a long and good experience with these courier services and also most researchers have their own accounts set up with them.

  • Is it possible to combine several orders in one shipment?

    Yes, but only if they all have been placed within a period of 3 weeks, if we already have the extra MTA for those clones in place, and if they don't exceed more than 100 ES cell clones in total.

Order

  • How long does it take to approve my account?

    It takes around 2 working days.

  • How is the procedure for the MTA?

    We require you to have read and accept HAPLOBANK Terms and Conditions. Please make sure that the MTA is fully signed, i.e. by a legal representative of your institute, and you or your supervisor as the recipient scientist. Once you have obtained all required signatures on the MTA, please send a pdf copy to office@haplobank.at. To save time, we strongly suggest you sending us a fully signed MTA as a pdf per email as opposed to a hardcopy.

  • I have created a HAPLOBANK account a while ago. My account is still not active.

    If your account is not active after 3 working days, please send an email to office@haplobank.at.

  • Searching the database, I found several clones for my gene-of-interest. How do I decide which clone(s) I should order?

    We would recommend selecting for the right clone(s) by considering following points:
    1. One gene trap insertion versus multiple gene trap insertions: We have clones in our collection that either contain only one (blue rows) or more than one insertion of gene trap cassettes (grey rows). Multiple insertions may mask the phenotype which you are interested in, so preferably you should select a ‘blue’ clone.
    2. In the column "Rel. Orientation" you will find information in respect to the orientation of the genetrap insertion relative to the gene. The genetrap is either inserted in "sense" or "antisense", and is thus disruptive and non-disruptive for your GOI, respectively. When the gene trap is flipped, the opposite scenario occurs; the gene trap becomes disruptive (when being non-disruptive before) or vice versa.
    3. Specified under the column "Mutation", you will find information regarding the location of gene trap insertion. If the mutagen is inserted in 1Intron, Intron, 5'UTR, CDS, and ncExon, it is disruptive. Ideally, your clone of interest carries an insertion in the first Intron (1Intron). Please be aware that clones that carry the gene trap in an intergenic region (i.e. annotated as Upstream, InterDown, InterUp), cannot be regarded as knockouts of the respective gene. Also, knockout clones that carry a mutation in the coding sequence are phenotypically not reversible!
    4. If you can choose between Tol2, Tol2GT, Retro or Lenti (specified under the column "Mutagen"), you should keep in mind that Tol2GT/Retro/Lenti are enhanced gene traps while Tol2 is a polyA-trap that carries EGFP! Please, check the relevant publications for details on each of the systems (Mayasari NI, Mukougawa K, Shigeoka T, Kawakami K, Kawaichi M, Ishida Y., Nucleic Acids Res. 2012 Jul;40(13):e97 and Schnütgen F, et al., Nucleic Acids Res. 2008 Nov;36(20):e133).

  • I have ordered ES cell lines some time ago. Can I check the status of my order?

    Yes, if you log in into your HAPLOBANK account and click on the button Order History.

  • I have not received any notification about my order. Is there any problem?

    As long as you received an order confirmation and sent all required documentation (i.e. MTA, import permits), your order is still in progress. Due to our preparation schedule, it can take up to 6 weeks for finalization and shipping of your order. You can check under Order History on the HAPLOBANK website the status of your order.

  • I placed an order yesterday and forgot to order another ES cell line. Can I add this cell line to the existing order or should I place an additional order?

    Unfortunately, we cannot modify an existing order, however, you are able to cancel it within 24h and place a new one. Alternatively, you can place an additional order and advise us via email to combine the shipment of individual orders. This is possible as long as individual orders are placed within 3 weeks and don’t exceed the limit of 100 ES cell lines in total.

  • Can I replace some ES cell lines, which are still pending with other ones?

    Unfortunately, we cannot modify an existing order.

  • Is there a way to save the content of the shopping cart and order at a later timepoint?

    Our website doesn’t offer shopping cart management, so you would need to save the Cell IDs in a separate file on your computer.

  • Can I come and pick up the cells directly at HAPLOBANK?

    Yes. After placing your order, please send us an e-mail and notify us. As soon as your order is finalized, we will contact you to make an appointment for picking up the cell lines.

Payment

  • Can I get a discount price if I order many ES cell lines?

    No. Please see our payment information.

  • My credit card is valid but was rejected during the payment process. Why?

    We have no possibilities to get detailed information about user transactions from financial institutes due to security reasons. Therefore, we recommend you trying again later and if it doesn’t work to check with your bank.

  • During the payment with my credit card the system asks for a password. What password?

    Your credit card is activated with the "Verified by Visa" or "MasterCard SecureCode" service which improves the security of online payment transactions. Therefore, you need a password to verify your credit card transactions. Please contact your bank.

  • I do not have a credit card. Can I pay in some other way?

    While we prefer payment by credit card, we are aware that in some institutions invoices can only be processed after the delivery of goods. If this is the case, please contact us prior to ordering, so we can change your access to the ordering system. After your order is placed, you will receive a Pre-Invoice with a PO number. Please note, that we charge 30 EUR processing fee for that.

  • What is a VAT Number in the registration form?

    The Value Added Tax, or VAT, in the European Union is a general, broadly based consumption tax assessed on the value added to goods and services. Every European Institution needs a VAT number for tax purposes.

  • I do not have the invoice/credit note for one of my orders anymore. Can you resend it?

    For your convenience, you can download the invoice/credit note in your HAPLOBANK account.
    Please follow these steps:
    1. Login with your user name / password.
    2. Select Order History on HAPLOBANK website and you will get a list of your orders.
    3. Click on the ’View’ button to display an order.
    4. Click on the ’PDF’ button beside the invoice ID to download the invoice.
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