FAQ Services GB

------------------------------------------------------------------------------------------------------------------------------

 

1. How do I obtain updates on the progress of my project of reproductive sciences?

We keep you updated on the progress of your project with a report at each stage: reception of your animals or animal products and materials on our premises, quality control reporting of cryopreservations, birth of pups, sending out biopsies for genotyping, confirmation of SOPF health status, dispatching of animals to your premises, etc.

If your require further information during the course of your project, you can send a request by email myproject@janvier-labs.com  or by telephone +33.(0)2.43.02.90.28. 

 

2. General assistance on reproductive sciences

The table below illustrates examples of the problems you may encounter with your strains and the solutions JANVIER LABS offers.

 

 

3. If I entrust my strain to JANVIER LABS to provide a service, do I lose my intellectual property rights on either the original strain or the pups produced? 

The animals, embryos or animal products and materials used by JANVIER LABS during the provision of services as well as their respective intellectual property rights, remain the property of the customer and under its sole control.

JANVIER LABS neither acquires any intellectual property rights nor in particular, the right to make use of or sell these elements.

 

4. The animals or animal products and materials required for the service will be provided by a colleague from another laboratory. Can you organize the transfer process? 

We manage all the logistics concerning the transfer process of your animals or frozen animal products and materials from anywhere in the world, including making contact with your colleagues, arranging transport, customs documents, etc.

We also ensure the delivery of your animals or frozen animal products and materials to anywhere in the world, either through our own transport service within Europe, or through specialised delivery firms in the rest of the world.

 

5. How are my animals housed at JANVIER LABS? 

Customers' animals are received at an external laboratory situated 8 km from JANVIER LABS SOPF breeding site and they are handled by employees working exclusively with non-SOPF animals. A the external laboratory, the animals are housed in ventilated cages and changed in a laminar flow hood to avoid any contamination between the strains from different customers.

After collection and initial washes, the embryos are transferred to the internal laboratory (8 km away) by a special team for being stocked or transplanted into pseudopregnant females. After transplantation, the recipient females are housed in isolators to ensure optimal management of health status of each strain.

This housing strategy, entailing the separation of infected animals and SOPF animals by successive shields, provides optimal SOPF status rederivation and breeding without the risk of contamination.

 

6. How can you help me if my colony has been infected by a pathogenic microorganism that is interfering with my results, when I need to finish my study as quickly as possible?

To get technical advice concerning your problem, contact us through myproject@janvier-labs.com. If you need 3-6 healthy males and 3-6 healthy females to get started, a standard rederivation would be the most appropriate solution. If you need a larger amount of animals to get started, then we would recommend a rapid colony expansion.  However, for a customised solution based on the genotype of your model and your specific needs, do not hesitate to contact us by telephone or email to discuss this with one of our experts.

 

7. Which infectious agents do you test for, relating to SOPF health status? Are you able to run additional tests? 

The SOPF health status is based on recommendations from FELASA who have drawn up a standard list of agents to test for, regarding SPF (Specific Pathogen Free) and SOPF (Specific Opportunist and Pathogen Free) health statuses. JANVIER LABS applies a health and safety policy in order to guarantee the maximum health and safety standard and is committed to providing high quality products for research (JANVIER LABS Health policy). All the analyses are carried out by independent laboratories, in order to ensure total objectivity in the results. Animals leaving our premises will have been tested for the following infectious agents (see table). 

If your strain (rat or mouse) is particularly sensitive to an agent that does not feature in this list, we can arrange for tests, accordingly. 

 

8. How are the health monitoring controls organised within your transgenic services department?

JANVIER LABS has the SOPF status health monitoring controls carried out by independent laboratories. For assisted reproduction services, 2 recipient mothers produced by embryo transfer are used as sentinels. These females are dispatched for being controlled 8 weeks after the embryo transfer. In the case of customised breeding, once the initial control has taken place, the sentinels are then dispatched every 13 weeks to periodically control the isolators maintained for a longer period (for a list of agents, cf FAQ 7).

 

9. In which circumstances it is necessary to opt for identification by genotyping?

For several services, such as rederivation and recovery, we offer you the possibility of identification by genotyping. This consists of marking the pups with transponders at 10 days old and taking a caudal biopsy of the litters produced. We send these biopsies to you so that you can proceed with genotyping and select the animals of interest you want to keep (you can also ask us to proceed with genotyping for you. In this case, you will receive a corresponding quotation). It is important to note that for rederivation and recovery with or without in-vitro fertilisation, there is a mendelian transfer of alleles outside of any transgenic influence. The genotypes of the males and females used should therefore be taken into account.

In which instance would this be necessary? The option of identification by genotyping is necessary if you are expecting pups with variable genotypes and for all analyses for which an identification animal per animal is necessary. For example, if a rederivation were done with heterozygote males from your strain and female wild types from JANVIER LABS, 50% of SOPF pups produced would carry the transgene. In such a case, to have the choice of 3-6 males and 3-6 females SOPF from the rederivation process where there are more than 10 rederived animals, it can be worth opting for this type of genotyping to optimize the number of animals of interest.

You need to take into account that we guarantee 3-6 SOPF males and 3-6 SOPF females, regardless of the genotype. According to which animals are provided or used, you need to estimate the transmission of the transgene to the progeny. In any event and as far as possible, (expected transmission of main transgene around 50%), we will manage to provide you with at least 2 carrying males, provided that the strain has no influence on the mendelian transfer of the genetic character in question.

The table below, broken down into different mating methods should assist you in your decision-making:

 

 

10. My strain has not been correctly maintained and will no longer be productive, given the age of the animals. Can you help me?

Yes, it is likely that we can save your strain, even if there is only one male or a few females remaining. We have considerable experience and several ways of rescuing lines where animals have stopped producing. Bearing in mind that the chances of success will always depend on the age of the animals, the quicker we intervene, the more likely we are to succeed. The rescue strategy will depend on the gender of animal that you have available (see the available technical sheet for strain rescue).

 

11. What is the Transgenic Services Project Sheet? 

Once you have accepted our proposition, we will send you a form to complete with our information requirements via email. This form will provide us with the details we need to set up your project (genetic and phenotypical characteristics of the strain, customer point of contact, type of service required, health monitoring controls, delivery addresses etc.) and to declare all the genetically modified organisms on our site to the appropriate regulatory bodies.

 

12. What is the average number of pups I can expect to obtain through embryonic transfer?

The graph below shows the average number of pups obtained after embryonic transfer coming from one of the following:

  • either directly collected embryos;
  • or frozen embryos;  
  • or embryos produced as a result of IVF treatment  (fresh or frozen sperm).

 

The process is to transfer a set number of embryos into the oviduct of pseudopregnant females, and then to count the number of pups produced. The result is expressed in terms of the number of embryos producing pups over the total number of transferred embryos, according to the different strains: Birth Rate (JANVIER LABS data taken from genetically modified embryos or from the JANVIER LABS catalogue)

 

 

13. Could you explain the 11-week delay between reimplantation and the delivery of SOPF animals? 

The SOPF animals of your strain will be delivered to you at the age of approximately 8 weeks, therefore ready for being mated.

We dispatch recipient females for a health check 8 weeks after their insertion in an isolator (the pups are 5 weeks old when their mothers are dispatched). We then have a 2-week period in which to receive the results of the health monitoring control and the validation of SOPF status. Once these results are received we schedule the delivery of the animals to your premises.    

The provision of the rederivation via embryo transfer service lasts a total of 11 weeks (D0 = embryo transfer  < D0 + 3 weeks = birth of pups < D0 + 11 weeks = delivery of SOPF animals)

 

14. I am unable to provide the number of animals required for the provision of a rederivation/cryopreservation service. Do I have any other options?

The number of animals required has been estimated by taking into account the fixed restitution objectives (the number of SOPF animals or number of straws of frozen biological material).

If you are unable to provide the number of animals required, we can adapt the programme in 1 of 2 ways:

  • By developing a colony in advance of the provision of the required service. This enables the maintenance of restitution objectives.
  • By reducing the restitution objective, which can be recalculated according to the number of available animals.

 

15. Why is it necessary to isolate a mature male a week before mating or collecting sperm?

The male (or dominant males) in a cage inhibits the synthesis of other males’ testosterone, therefore reducing their fertility. We have to therefore isolate males for at least a week in order to avoid this phenomenon and to reestablish the process of producing spermatozoids.

 

16. Is it possible to decontaminate a strain by using fresh or cryopreserved sperm?

Rederivation through sperm is possible and often practiced, but it does have to follow steps in a precisely managed procedure. Indeed, it should be noted that a number of agents such as viruses can be detected in sperm (for example, Agca et al.2007 Janus et al., 2009). For this reason, the direct insemination of a female (non contaminated) with this sperm will not be able to decontaminate the strain in question for those infectious agents.

Therefore the plan would be to manage the decontamination though embryo transfer using in vitro fertilisation. Indeed, the embryo’s pellucide zone forms a barrier which is impenetrable by the main contaminators and so decontamination takes place by the successive washing of the embryos and the transfer of them into non-contaminated surrogate females (for example : Suzuki et al., 1996 ; Van Keuren and Sanders 2004). Research carried out by Janus et al., 2009 and Mahabir et al., 2007 showed that it is more difficult to eliminate certain viruses (notably, the MMV) and that conditions need to be more tightly managed. At the same time, these researchers who drew attention to these risks (Mahabir et al., 2009 ; Mahabir et al., 2008 ; Peters et al., 2006) also proved that even for these viruses, decontaminations via IVF can be efficient. The following steps need to be taken in the process of decontamination via fresh or frozen sperm, to ensure that the decontamination is efficient (at each step, different barriers and limited zones need to be defined):

  • Collection or defrosting of sperm from the strain that needs decontaminating;
  • Collection of ovocytes from donor females (regardless of the health status of these females) and in-vitro fertilization with sperm from the strain to be decontaminated;
  • Culture for 24 hours to obtain embryos with stage 2 cells
  • Washing 10 times minimum (IETS recommendations)
  • Transfer into pseudopregnant females with known health status (for example, SOPF);
  • Birth of pups and breeding in conditions that maintain the health status. These animals now have the genetic characteristics from the strain where the original sperm and female donors of ovocytes came from, with the health status of the surrogate mother (SOPF).

 

17. Your restitution objective after rederivation via embryo transfer is 3 to 6 SOPF males and 3 to 6 SOPF females. How do you identify the restituted animals?

We restitute up to 6 SOPF males and 6 SOPF females of your strain, regardless of their genotype. If, after the embryo transfer, we obtain more than 6 males and 6 females of your strain, we can, if necessary, offer you the possibility of identification (see FAQ 9), which consists of identifying the animals (with a microchip) and dispatching biopsies for genotyping to you. This would allow you to choose the most appropriate animals to develop your colony on your premises. This option is invoiced at the current rate.

 

18. How should I receive the pregnant females coming from the treatment of rapid decontamination via embryo transfer, on my premises?

The pregnant females (about 15 days old) will be delivered to you in one parcel with the date they are expected to give birth. Upon reception, they should be separated and nest-making material should be provided in their cages.

The housing should not be changed before the birth date in order to avoid the risk of abortion or cannibalism.

 

19. What factors should be taken into account to decide whether my strain should be cryopreserved by sperm or embryos?

The choice of cryopreservation method is based on the knowledge and analysis of the characteristics of the strain in question. A number of constraints imposed by cryopreservation techniques should be taken into account.

Indeed, when the sperm is cryopreserved, only the genetic heritage of the male haploid is preserved. Revitalisation requires fertilisation (in-vitro) with, generally, females with the same genetic base but with wild genotypes for genetic modification. The individuals produced are necessarily heterozygotes for genetic modification. For this reason, management of a more complex strain (several mutations) is more complicated and risky with this technique. In fact, there is a separation of different transgenes during recovery, according to the genotype of males used for the cryopreservation. However, the cryopreservation of sperm is very simple and rapid, less costly and potentially producing a greater number of animals through recovery than through the cryopreservation of embryos.

The advantage of cryopreserving embryos is that it allows the saving and revitalising all of the genetic characteristics of the strain (what is cryopreserved is obtained during recovery). It is for this reason that this technique is chosen to manage genetic derivation and the cryopreservation of complex strains (multiple mutations and the maintenance of a particular genetic base).

JANVIER LABS advises you to analyse the characteristics of your strain in order to decide whether to cryopreserve sperm or embryos. If you so wish, we could do this analysis together and assist you in the most suitable choice for your needs

This table summarizes the elements to consider in the selection between sperm and embryo cryopreservation. We can assist you for the selection. Please contact us at myproject@janvier-labs.com.

Criteria

Sperm cryopreservation with(JAX™ Sperm Cryo Kit)

Embryo cryopreservation

Genetic material to be cryopreserved

Haploid

Diploid

Number of transgenes

1-2

Several

Genetic background

Inbred or
not so important

Original

Multifactorial genotype
(genetic interaction)

Controlled

Suitable for any model

Do-it-yourself

YES

NO

Genotype management at the first generation of recovered animals

Heterozygous

Depending on the cryopreservation(homozygous)

Quantity management
at recovery

+++

-

Cryopreservation cost

Low

High

Quantity, genotype and cost management

+++

+

Strain use

?

?

 

JAXTM is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

20. Why does JANVIER LABS cryopreserve embryos using vitrification and not slow-freezing?

The technique of embryo cryopreservation reliably exists since the beginning of the 1980s. At that time the method used was slow-freezing (also known as the equilibrium method). It consists of placing the embryos in a cryoprotective environment and gradually lowering the temperature. This procedure enables the embryos to be frozen whilst avoiding the formation of ice crystals in the cells, which could change or destroy them. Vitrification (also known as the non-equilibrium method) consists of placing the embryos in a highly confined cryoprotective environment in order to avoid ice formation outside of the cells leading to an efflux of intracellular water. The embryos are then immersed in liquid azote. This rapid freezing enables the embryo to be set in what can be called its vitreous state, given that the water has not had the time disorganise itself into ice crystals. 

Efficient vitrification procedures have been developed over the past few years because the method is easier and quicker (no need for equipment to gradually bring down the temperature as in the slow-freezing method). It appears that this technique is less likely to aggress and alter the cell, resulting in being less invasive and disturbing in the intracellular environment. For example, this technique offers solutions for the cryopreservation of ovocytes that are more efficient than slow-freezing.

Here are a few references that give a little insight into the vitrification method and its efficiency concerning the cryopreservation of ovocytes and preimplantation embryos: Nakao et al., 1997 ; Zander-fox et al. 2013 ; Vanderzwalmen et al. 2013 ; Kohaya et al., 2013 ; Nakagata et al., 2013 ; Rezazadeh et al., 2009 ;

On this basis, JANVIER LABS has developed a successful protocol and service for the cryopreservation of embryos using the vitrification method. Therefore we recommend the cryopreservation of your strains by this method. We also use vitrification to provide you with frozen sperm from our JANVIER LABS strains at each stage of preimplantation development, notably to be used for changing the murine genome (for example Quickblasto®).

 

21. Concerning the embryos stocked in straws then frozen, are they firstly decontaminated from any possible germs present in my strain?

YES, straws are filled up once the embryos have been washed in a sterile environment and within a microbiological health security unit.

 

22. What percentage of viable embryos can I expect from the revitalisation of embryos?

The graph below shows the percentage of viable embryos at the 2-cell stage at the time of defrosting the embryos previously frozen by JANVIER LABS according to their genetic base (in blue). These embryos are put in a culture to ensure their recuperation of biological activities until the 4-cell stage (in red). As a general rule, for 100 embryos defrosted, 60 to 90 of them are still alive (through observation with binocular magnifying glasses) and 50 to 70 % of these embryos develop to the 4- cell stage (JANVIER LABS data from lines of genetically modified mice treated by JANVIER LABS). To estimate the number of pups that we can obtain (birth rate), the viable embryos need to be transferred to surrogate females (see FAQ 12).

 

23. How effective is an IVF after cryopreservation with the JAX™ Sperm Cryo Kit?

The Jackson Laboratory designed the JAX™ Sperm Cryo Kit based on a study published in the Plos One Journal (Ostermeier et al., 2008). The efficiency of a sperm cryopreservation is evaluated by the sperm fertilizing ability at recovery using IVF. The summary of the results published in the study is presented below as a percent of fertilized oocytes reaching the two-cell embryo stage. To be able to estimate the number of pups (birth rate), the embryos obtained from the IVF will be reimplanted into recipient females. (See FAQ 12)

JAX™ is a registered trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

24. What are the benefits and advantages of the double service provision: cryopreservation (sperm or embryos) and decontamination through embryo transfer?

This double service provision allows us to ensure not only the rederivation of a strain but to also secure a back-up in frozen form.

An in-vivo quality control of the frozen embryos allows for the restitution of SOPF individuals of the line. 

Decontamination through transfer of naturally produced embryos will stimulate and allow for the fertility of males, whose sperm will be cryopreserved later, to be checked.

 

25. How to choose and interpret quality control checks for the freezing of sperm and embryos?

Cryopreservation of germoplasm is only of interest (generally speaking) if it is possible to efficiently get back viable individuals with the required genetic characteristics. Moreover, building up a stock of frozen material (sperm bank or embryos) does not imply recovery in the short-term. It is therefore indispensable to ensure that the material has been correctly frozen. For this reason, quality control checks of the freezing are necessary. This check will consist of controlling the viability of the cryopreserved material with maintenance of their biological activities and functions. Quality control is also useful to provide necessary data for an efficient future recovery.

 

Quality controls (QC) for sperm cryopreservation

This table summarises the advantages and disadvantages for each quality control check.

  

Quality control

Purpose / Procedure

Advantages

Disadvantages
(evaluation of risk
)

Security 
of QC

JANVIER LABS Advice

Sperm analysis at thawing

 - Thawing of one straw

 - Measurement of sperm mobility, mortality and density

 - Rapidity

 - Cost

 - Necessary to analyse the IVF data

 - No measure of the sperm biological function i.e. fertilization.

 - No estimation of the transgenes effect on the efficiency of the cryopreservation

 - Incomplete recovery data

Low to medium

Insufficient

in vitro fertilization (IVF): in vitro recovery rate

Estimates the sperm fertilizing ability after thawing

 - Thawing of one sperm straw

 - Fertilization of fresh oocytes by thawed sperm

 - After 24h of incubation, estimation of the number of fertilized oocytes at 2-cell stage

 - Results expressed in % of 2-cell stage embryos 

 - Measurement of the maintenance of the biological function

 - Main recovery data

Partial estimation of the transgene effect on the cryopreservation

High

Yes

(see QC1 « In-vitro »)

Animal births:

birth rates

Estimates the ability to produce animals after IVF:

 - IVF quality control

 - reimplantation of the divided embryos into pseudo-pregnant females

 - Results expressed in % of pups born

 - Measurement of the biological function

 - More detailed evaluation of the transgene effect

 - complete recovery data

 - Longer and more expensive

 - the level of influence of the transgene has to be estimated depending on the model

Very high

Depending on the strains

(See QC2 « in-vivo »)
we recommend to genotype the pups if necessary

 

How should I analyze a quality control for sperm cryopreservation?

An in vitro quality control (QC1) indicates the number of oocytes fertilized and developed to 2-cell stage after IVF. The table of FAQ 23 shows the average IVF rate per strain cryopreserved with the JAX™ Sperm Cryo Kit.

Based on the percentage of 2-cell stage embryos and according to the genetic background and the transgene, the quality control results will be subdivided into the following category:

  • Re-freeze
  • Recovery feasible
  • Fair
  • Good
  • Excellent

 

When the recovery will be required and in order to obtain enough pups, a larger number of oocytes will be used if the quality control result was “recoverable” than if the result was good or excellent.

Therefore, a quality control is very useful as it provides information on the recovery output (especially the number of oocytes). Regarding the transfer of the embryos from the quality control, 40 to 50% of them will produce pups (See FAQ 12).

Example: Cryopreservation of a genetically modified strain on a C57Bl/6JRj background and a quality control result at 25%. The result is considered “good” and confirms the cryopreservation was successful.

When you will decide to recover your strain to re-establish a colony, you will need about 3 to 5 breeding pairs.

On the embryo transfer chart (See FAQ 12), the C57Bl/6JRj strain almost reaches a 50% birth rate: namely 50 pups obtained on 100 re-implanted embryos (be careful, the transgene can affect this result). Here are your recovery data:

  • A 10µ-drop from a thawed sperm straw and IVF with 80 oocytes
  • Over 80 oocytes, only 20 will develop to 2-cell stage (QC1 with a 25% rate)
  • The re-implantation of these 20 embryos into pseudo-pregnant females will produce 10 pups (thus 50%)

 

The in-vivo quality control or QC2 indicates the number of pups obtained after IVF from frozen sperm. In this case, we have all the recovery data available and we can readjust the conditions later at recovery. Indeed, for this quality control, we know the number of pups to expect based on the number of embryos obtained from the IVF. However, depending on the genotype of the males used for cryopreservation, you might need to evaluate if the genotyping of the pups is necessary in order to be sure that they carry the transgene(s) and are not wild type. (See FAQ9)

We can re-use the above example but the 50% rate in embryo transfer is already expressed by the QC2 and therefore takes into account the eventual effects of the strain genetic modification.

 

Quality controls (QC) for embryo cryopreservation

This table summarises the advantages and disadvantages for each quality control check.

  

Quality control

Purpose / Procedure

Advantages

Disadvantages (evaluation of risk)

Security  of QC

JANVIER LABS Advice

Embryo analysis
at thawing

 - Thawing of one straw

 - Observation of viability

 - Rapidity

 - Cost

 - No measure of the biological function i.e. embryonic development.

 - No estimation of the transgenes effect on the efficiency of the cryopreservation

 - Incomplete recovery data

Low

Insufficient

in vitro culture

After freezing, measurement of the capacity of embryos to develop within a 24-hour culture:

 - Thawing of a straw containing embryos

 - Observation of embryos during thawing: number of viable embryos out of total number of thawed embryos (expressed in %)

 - 24-hour culture in vitro

 - Result in % of embryos at the 4 to 8-cell stage

 - Measurement of the maintenance of the biological function at the first preimplantation stages

 - Partial data on recovery aims

Partial estimation of the transgene effect on the cryopreservation

High

Yes

(see QC1 « In-vitro »)

Animal births:

birth rates

After freezing, control of the embryos' capacity to become animals:

 - reimplantation of embryos into pseudo-pregnant females

 - Results expressed in % of pups born

 - Measurement of the biological function (all development including the pregnancy period) 

 - more detailed evaluation of the transgene effect

 - complete recovery data

 - Longer and more expensive

 - Complete evaluation of the transgene up until birth

Very high

Depending on the strains

(See QC2 « in-vivo ») we recommend to genotype the pups if necessary

 

How should I analyze a quality control for embryo cryopreservation?

The QC1 for the cryopreservation of embryos provides information on the efficiency of the cryopreservation with a return to embryonic development. We consider that a QC is validated if it is in excess of 50% (50% of embryos frozen at the 2-cell stage have developed to the 4 to 8-cell stage). This therefore validates the cryopreservation, but it should be noted that on average, JANVIER LABS is between 70 and 80% (see FAQ 22).

When it is necessary to carry out a recovery, the embryos will be transferred into a pseudopregnant female directly after (same as for a QC2). If a QC2 has been done, this data can be directly used; if a QC1 has been done, the guarantee of development allows us to ensure recovery criteria, given that 70 to 80% of embryos are viable and that 50% of these embryos will produce pups (see FAQ 12).

For example, if I wanted to revitalise 10 pups to relaunch my line:

  • I thaw around 25 embryos and I will obtain 20 viable embryos (80%)
  • I transfer these 20 embryos and I obtain around 10 pups (50%)

 

JAX™ is a trademark of the Jackson Laboratory registered in the United States. All rights reserved.

 

26. What is the time limit for the use of the JAX™ Sperm Cryo Kit?

Only the cryoprotective medium is perishable. The medium is delivered along with the Kit and has to be stored at -80°C upon receipt and until use.

Upon delivery, the medium has an 12 to 16-month duration of use maximum.

JAX™ is a trademark of the Jackson Laboratory registered in the United States. All rights reserved.

 

27. How should I store the JAX™ Sperm Cryo Kit?

Upon receipt, the cryoprotective medium has to be stored at -80°C and the other components have to be stored at room temperature. Please make sure to keep all the content of this Kit as everything will be used for the cryopreservation and especially the StyrofoamTM freezing box and raft which are essential during the process.

JAX™ is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

28. What are the required skills to cryopreserve strains on my own using the JAX™ Sperm Cryo Kit?

There are no particular skills required to do the cryopreservation on your own using the Jax™ Sperm Cryo Kit. You just need to have experience in dissection and biopsy sampling from mice and basic laboratory skills. However, you need to practice first, especially for dissection of vas deferens and cauda epididymis which is a key step to succeed the cryopreservation. Moreover, the Kit contains a batch of straws labelled “practice” for this purpose.

JAX™ is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

29. Which IVF technique one should use if the cryopreservation was performed with the JAX™ Sperm Cryo Kit?

If you have the required skills to perform IVF and if you want to do your quality controls yourself or even the recovery of your strains, it is possible on the condition that you have all the required elements and IVF protocols to use. The Jackson Laboratory designed this Kit to simplify and optimize the IVF and recommends the technique published in the following article (Ostermeier et al., 2008).

There is an alternative IVF technique developed by the N. Nakagata group (Takeo et Nakagata, 2011). This technique is more complex, more expensive and requires some adjustments. However, it is more effective in certain conditions.

If we consider the different steps of a sperm recovery and the variation of results due to the recipient females’ pregnancy, we recommend the simple technique published in 2008 (Ostermeier et al., 2008). Indeed, obtaining mice to start out a colony using one method or the other will be fairly similar.The Kit is compatible with the use of the IVF technique described by Nakagata (Nakagata et al., 2014). 

If you need any advice on the different IVF techniques available to recover sperm cryopreserved with the JAX™ Sperm Cryo Kit, please contact us at myproject@janvier-Labs.com.

JAX™ is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

30. Can I use the JAXTM Sperm Cryo Kit to cryopreserve sperm from all mouse strains and other mammalian species? 

The JAXTM Sperm Cryo Kit is optimized for the use on mouse sperm but is not designed for sperm cryopreservation of other mammalian species. Sperm from all currently tested mice strains can be cryopreserved, however the in vitro fertilization rate after thawing is different from one strain to another (Ostermeier et al., 2008, see figure of FAQ 23).

JAX™ is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

31. How long can I keep the sperm or embryo in liquid azote for?

The cryopreservation of laboratory mice biological material began over 40 years ago and today it is widely practiced in institutes. (review: Mo Guan et al., 2012). Having the knowledge and a real perspective on the storage time in liquid azote is therefore essential. If we consider that the cryopreservation of sperm or embryos has been correctly carried out, it is theoretically possible to keep them indefinitely. On the practical side, we have some 15 years of hindsight for sperm cryopreservation and over 30 years for the cryopreservation of embryos (Glenister et al.,1984 ; Kaneko et al., 2006, Leibo et al., 1994 ; Riggs et al., 2010).

 

32. I have a straw of mouse frozen sperm. Can you recover the strain and ship the live mice to me or to my collaborator? Is one straw of sperm sufficient to obtain mice and start out the colony?

Yes, we can recover the strain if the sperm was frozen using the JAXTM Sperm Cryo Kit and if you can provide an acceptable quality control result (namely more than 5%). One frozen sperm straw is enough to obtain 3 to 5 breeding pairs.

If an alternative method or no quality control result is available we attempt to revitalize. At least two straws will be required in this case.

JAX™ is a trademark of The Jackson Laboratory registered in the United States. All rights reserved.

 

33. How can I send you straws of sperm or frozen embryos (for storing or quality control, for example)?

To send your straws to our premises, our own transport service of specialised transporter for frozen biological material can deliver and collect from a ready-to-use dry shipper.

 

34. I wish to obtain a strain belonging to a colleague in another institute and whose straws of sperm or embryos are being stored at JANVIER LABS. How should I proceed?

In order to obtain a strain belonging to a colleague, he will need to give us his written approval, either by email or using a form that we provide to approve the release of biological material, giving precise details of the transfer (name of strain, name of colleague, number of straws etc.).

 

35. I have straws of sperm or embryos being stored at JANVIER LABS. What should I do when the storage reaches its term?

Just before the end of the storage period, you will be informed of the finishing date by email. The storage period can be prolonged at your request.

If needed, we can send you an estimate for the prolongation of the storage period.

 

36. What are the storage conditions of my straws of sperm or embryos at JANVIER LABS?

The straws of sperm or embryos from our customers’ strains are totally immersed in liquid azote on two independent sites 8km away; the geographical distance is to guarantee you a high level of security for your biological material.

An alarmed control system assists us to monitor the cryostorage.

 

37. Is it complicated to use Quickblasto® for my ES cells injections?

No, a user guide manual is provided with kit, which is easily understood by anyone having already some experience with manipulating embryos at the preimplantation stage.

 

38. How do I estimate the number of straws to be thawed in order to have enough blastocysts for my ES cells injections?

Quickblasto® gives you blastocysts in which to transfer embryonic stem cells. The table below illustrate the average performance registered at each stage for the 2 most currently used stems for the creation of genetically modified mice using Quickblasto®.

From this data, injections can be scheduled and notably the number of straws/embryos to be thawed can be estimated:

For example: if you wanted to obtain 6 chimera mice by cloning using blastocysts C57BL/6NRj:

  • Litters should consist of around 10 pups (of which 60% chimera mice).
  • For this result, you need to have transferred around 30 blastocysts into pseudopregnant females environ 30 blastocystes (31.8% birth rate): we recommend transferring 14 blastocysts (bilaterally) per pseudopregnant female, i.e. 2 females in our example.

 

You will need to thaw around 50 embryos, i.e. 2 straws to obtain around 30 blastocysts to be transferred (67.4% development rate. You should allow for some loss during the injections).

 

39. How do I obtain updates on the progress of my project of customised breeding?

For all customised breeding projects, a client interface (AniBio) allows to visualize all animals (http://anibio.janvier-labs.com/Default.aspx). This interface does not require any specific set up (besides an internet connection) and is available 24/7. With this interface, you can directly give your instructions (mating, euthanasia, shipment of animals...). Once you have carried out your genotyping analysis, you can assign genotype of your animals yourself. Thanks to this tool, you have a traceability of all your colonies, of your requests for associated services and you can have a real-time cost estimate.

If your require further information during the course of your project, you can send a request by email myproject@janvier-labs.com  or by telephone +33.(0)2.43.02.90.28. 

 

40. What kind of customised breeding do you offer according of the genotype of my animals?

JANVIER LABS offers customised breeding of transgenic rodents; this can consist of marking animals and taking biopsy in order to allow genotyping of transgenic animals. We send these biopsies to you so that you can proceed with genotyping or JANVIER LABS can arrange genotyping for you.

The table below presents the different customised breeding offers for rodents (rats and mice) we propose:

 

SPF breeding in IVC

(Individual Ventilated Cages)

SOPF breeding in isolator

Services

Breeding in individual
ventilated cages

Breeding in isolator

SPF health monitoring controls

SOPF health monitoring controls

Mating

Weaning

Colony management via
a dedicated customised breeding software (AniBio)
(24/7)

Follow-up and scientific support of the project by a dedicated project manager

Options

Repatriation of animals

Rederivation of strain

Identification (transponder, ear or other)*

Biopsies (tails or ears)*

Genotyping by PCR, establishment of a genotyping protocol

Shipment of animals depending on needs (age, sex, genotype)
based on predefined batches or according to availabilities

 

* In case of identification, animals are marked by transponder (electronical microship) and biopsies are carried out. This system can be used if you do genotyping animals or if we proceed with genotyping for you. It is important to know that animals can be marked from 10 days old. This allow to have genotyping results before weaning and to keep animals of interest only and this reduces the number of breeding cages.  

 

 

41. For customised breeding, how can I know the quantity of pups born / weaned, date of birth of pups, gender...?

All these information are available in the customised breeding software (AniBio).  (http://anibio.janvier-labs.com/Default.aspx).

 

42.  How can I know if my request has been taken into account in AniBio?

All your requests can be followed up in the customised breeding software AniBio (date of the request, animals concerned, staff who works on your request, status of the request, date of realisation...).

http://anibio.janvier-labs.com/Default.aspx).

 

43. Is is possible to carry out protocols on my animals in customised breeding?

Yes, you can send all your requests (for exemple: blood sampling, organ sampling, surgery...) Contact us at myproject@janvier-labs.com. We will send you a quotation according to your needs.