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This time let's share Frequently Asked Questions (FAQ) for Transgenic Services; b' c8 A) s0 t% R

5 K9 I3 ~# i5 C: o! {! \7 k3 H9 TFrequently Asked Questions (FAQ) for Transgenic Services
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: i5 q( l8 c- {3 L( i* w+ CWhy should customers choose Cyagen’s transgenic services?( n( U4 D3 j( F" i' a: G1 K

) R7 t( Q! H- a3 @: l7 l& J+ QCyagen Biosciences is a U.S. company committed to providing high-quality products and services to life scientists around the world at favorable prices. We have a subsidiary in Guangzhou, China, that offers transgenic services. Customers choose our transgenic services because:
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1. Our highly experienced experts provide top-quality services with consistency and rapid turnaround.
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& x  R5 u. X5 D% G2 s( I4 b* n2. We offer exceptionally affordable prices that beat all our competitors.
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What transgenic services does Cyagen offer?  
2 [$ u7 s+ |% r  L; R2 S3 a1. We produce transgenic mouse embryos or live transgenic mice using either plasmids or BACs.
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2. We produce knockout mice using knockout ES cells.* `& u  z% p/ w  N! ]( i

6 }! M; C6 B$ i; D8 Z; w/ ^( z; j  {9 }3. We obtain knockout ES cell clones using targeting constructs per customers’ specification.
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& _8 p: Z9 v' t1 `4. We build DNA constructs for making transgenic or knockout mice per customers’ specification.
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5. We perform genotyping assays per customers’ specification.0 z: M1 `9 R8 i; {

/ z7 h) s( v! y0 g; u" S1 @1 z" O- ^6. We perform LacZ staining on transgenic embryos or tissues.
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7. We re-derive specific pathogen free (SPF) mice from non-SPF mice for customers.
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% ]1 x* }- x% M( J. _1 V8. We cryopreserve mouse embryos for customers.
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$ \0 \* Z" J9 UWhat does it mean to produce transgenic mouse embryos?
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6 k/ [7 r8 P3 U  xMany of our customers do not need live transgenic mice. Rather, they want to obtain transgenic embryos at various stages of gestation to study the behavior of the transgene during development. Typically, they create DNA constructs (either plasmids or BACs) in which the LacZ reporter gene is placed under the control of enhancer/promoter elements of interest. Transgenic embryos made from these constructs are dissected out of surrogate mothers and stained for LacZ activity, in order to reveal how the enhancer/promoter elements drive gene expression during embryonic development.* {- e! {+ V2 T1 m* T
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How many embryos are injected for transgenic production and what is the success rate?
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Transgenic embryo production is carried out in sessions, with around 200 embryos injected per session. For plasmids, 3-4% (5-8) genotype-positive embryos are typically obtained per session. For BACs, 1-2% (2-3) genotype-positive embryos are typically obtained per session. These success rates are on par with or better than other transgenic service providers. Because the success rate for BACs is about half of that for plasmids, we recommend that our customers purchase one session for each plasmid construct and two sessions for each BAC construct. Discount is available for the second session of a construct. For the production of live transgenic mice, the number of genotype-positive animals obtained per session is somewhat lower than that for transgenic embryo production, because some embryos are lost at birth (for example, the surrogate mother sometimes eats the newborns)., ]+ _% G! c3 T4 o$ q; Y6 c  h

. B# m- w& n: O0 ?+ b* HIs it necessary to linearize DNA in making transgenic mice?
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Researchers have traditionally linearized both plasmids and BACs before injection. However, a growing number of labs no longer linearize BACs for injection. They found that circular BACs achieve similar LacZ staining patterns in transgenic embryos as their linearized counterparts. This has indeed been our own observation. We therefore recommend that our customers consider injecting circular BACs, which saves considerable amounts of effort in DNA preparation and is also likely to lead to better results (higher frequency of transgenic embryos) since circular BAC is easier to inject. We also stress that the quality of the DNA is paramount to the success of the project. We will provide customers with detailed protocols for DNA preparation when initiating a project.
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When producing transgenic embryos, what fraction of genotype-positive embryos is LacZ positive?  9 I* W1 t! m+ k0 \+ T
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This depends on the biology of the construct. For some constructs, none of the genotype-positive embryos are LacZ positive even after many genotype-positive embryos are generated. For other constructs, all the genotype-positive embryos are LacZ positive. There are also constructs for which some genotype-positive embryos are LacZ positive and some are not. Generally, plasmids produce more variable LacZ staining, meaning that genotype-positive embryos made from the same plasmid can have greater variability in the presence/absence of staining, the patterns of staining, and the intensity of staining. In contrast, BACs produce more consistent LacZ staining, meaning that all the genotype-positive embryos tend to have similar staining outcome. This makes sense because BAC expression is less dependent on integration site than plasmid. If a construct has generated several genotype-positive embryos that are all LacZ negative, then it generally means that the lack of LacZ expression is due to the biology of the construct.
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8 j5 y5 {; G: M4 bWhat procedures does Cyagen follow for fixing/staining transgenic embryos?
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We have performed successful fixation and LacZ staining of transgenic embryos spanning a range of ages, from E8.5 to just before delivery. We use Millipore’s kit and accompanying protocol for fixation/staining. For older embryos, typically E15.5 or older, we cut them into several parts (with customers’ approval) before fixation/staining to ensure the proper penetration of chemicals.
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Is it possible to customize procedures for fixing/staining transgenic embryos?
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3 h% z- |" {) I$ X2 v3 UYes. We can follow custom protocols provided by customers when fixing/staining embryos. For example, most customers have us perform LacZ staining but some customers want us to only fix the embryos so they can do the staining themselves. It is also common for customers to request the dissection of specific organs for LacZ staining.& ~. E& Z3 c  Y, K2 H

- [, m  m' P" K) r; [What mouse stains does Cyagen use?
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3 X  [. n- f$ ^1. For producing transgenic embryos or transgenic live mice, we typically use FVB/N embryos for pronuclear injection. Upon customers’ request, we can also use embryos of other strains. We use CD1 females as surrogates.
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2 N% t1 w, y! I) d; r2. For producing knockout mice, we typically use B6xB6D2F1 embryos for blastocyst injection. We use CD1 females as surrogates.
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) {* Y9 w0 d, E+ u; K# AWhere does Cyagen source its animals?) H7 o* Y  [! U3 F' d4 J
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We source our animals mainly from Charles River Laboratories. Currently, only mice from Charles River, Jackson Labs and Taconic Farms are allowed into our facility.
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- j0 `4 w9 v- a4 [# p7 W/ WHow are Cyagen’s mice housed?
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All mice are housed in individually ventilated cages (IVC) in a state-of-the-art ultra-clean SPF barrier facility. Our facility meets and often exceeds animal health and welfare guidelines.
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8 K: I+ l1 q5 Q' ?) {7 BHow do customers ship DNA to Cyagen?8 r" ^3 x! E$ d& s+ Y0 K
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Customers can ship plasmid or BAC DNA to us by an express carrier under ambient temperature. In our experience, DNA quality is not compromised by this shipping method if the DNA is prepared according to our recommendation. We will test DNA quality before injection and inform customers of any signs of quality problem./ |6 v$ V$ j5 ^0 d; A# `/ ^
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How does Cyagen ship embryos to customers?' F- R3 r  `! E: K
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We ship embryos under ambient temperature by express carrier. It typically takes 3 work days to reach anywhere in the world. This has worked well for all our customers, whether they have us do LacZ staining or perform their own staining after receiving fixed embryos from us.
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9 U' z5 R* A3 H8 \What is the turnaround time? ; b/ `* A) E! q- ~3 z
1. For transgenic embryo production, the typical turnaround time from receipt of DNA to shipment of embryos is 4-5 weeks (1-2 weeks for pronuclear injection, ~2 weeks for gestation, and ~1 week for processing of embryos including dissection, fixation, LacZ staining, extraction of DNA from yolk sac, and genotyping).
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( q1 e! t7 n9 T2. For live transgenic mice production, the typical turnaround time from receipt of DNA to shipment of mice is 8-9 weeks (1-2 weeks for pronuclear injection, ~3 weeks for gestation, and 3-4 weeks for weaning and delivery).' ~7 n* |1 V5 S

' d) \8 f# U. B3 YHow does Cyagen protect customers’ IP?9 O. f* D: M5 |, i3 z& a6 q
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All materials from our customers, such as DNA constructs, PCR primers, and cells, are coded internally once they reach us, and kept strictly within the company’s premises. All information and experimental results of customers’ projects are maintained in secure places and kept apart from all other projects. Only authorized personnel have access to these materials and information.

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