DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Claims
Claims 1-4, 6-7, 9-14 and 18-19 are currently pending.
Claims 1 and 18 are amended.
Claims 5, 8, 15-17, and 20 is cancelled.
Claims 1-4, 6-7, 9-14 and 18-19 have been considered on the merits.
Withdrawn Rejections
The rejection made under 35 U.S.C. 112(b) is withdrawn in light of the clarifying amendments submitted on 12/16/2025.
New and Maintained Rejections Necessitated by Amendment
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-4, 6-7, 9-14 and 18-19 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 1 and 18 contain the phrase “forming a medium packed monolayer of Drosophila embryos with minimal overlap”, which is indefinite. The terms “medium packed” and “minimal overlap” in claims 1 and 18 are relative terms which renders the claim indefinite.
The term “medium packed” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification states “[i]n one exemplary embodiment, a medium packed monolayer of embryos can occupy about 30% of the total mesh area” ([0152]), however does not provide an explicit definition of “medium packed” to mean specifically about 30% coverage of the total mesh area. It is not clear if the term “medium packed” is meant to limit to, for example, around 50% packed, a range of between 30-70% packed, or about 30% packed as mentioned in the specification. Thus, appropriate correction/clarification is required. Applicant may amend claims 1 and 18 to include that the monolayer of embryos occupies about 30% of the total mesh area as recited in the specification, if desired.
The term “minimal overlap” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification states “[i]n one embodiment, after permeabilizing, a brush may be used to break up clumps into individual embryos floating as a monolayer with minimal overlap” ([0145]), however does not provide an explicit definition of “minimal overlap”. It is not clear if the term “minimal overlap” is meant to limit to, for example, essentially no overlap, or 0-20% of embryos overlapping, or under 50% of embryos overlapping. Thus, appropriate correction/clarification is required. Applicant may amend claims 1 and 18 to include that the method steps require brushing the clumps of embryos after permeabilizing to break embryo clumps into individual embryos with no overlap as recited in the specification and demonstrated in Fig. 10B, if desired.
Claim Interpretation
Claim 1 has been amended to include the phrase “with the embryos forming a medium packed monolayer of Drosophila embryos with minimal overlap”.
The term “medium packed” has been rejected under 112(b) above, for being a relative term which is not explicitly defined by the specification. The term “medium packed” is being interpreted to mean anywhere between about 30-70% packed, or in the alternative about 50% or half packed.
The term “monolayer” is not explicitly defined in the instant specification. However, the specification states that “In one exemplary embodiment, a medium packed monolayer of embryos can occupy about 30% of the total mesh area” ([0152]). Therefore, the term “monolayer” is being interpreted to mean a single layer of embryos, in which the embryos can be either sparse or densely packed next to one another.
The term “minimal overlap” has been rejected under 112(b) above, for being a relative term which is not explicitly defined by the specification. The term “minimal overlap” is being interpreted to mean a majority of the embryos do not overlap.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 6-7, 9-14 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Gilad et al (US20230020006A1)(reference of record), effectively filled 12/10/2020, as evidenced by Kuntz et al (PLoS Genet, 2014)(reference of record), in view of Mazur et al (Academic Press, 1993) (reference of record), and Steponkus et al (Cryo-Letters, 14, 375-380, 1993) (reference of record).
With regards to claims 1 and 18, Gilad teaches a method of cryopreservation of Drosophila embryos ([0046]) including the steps of collecting embryos, staging, dechorionating, permeabilizing, loading with cryoprotective solution (CPA), dehydrating ([0051]), transferring to a cryomesh, and placing in a cryogenic coolant as required by claims 1 and 18 ([0013]). The dehydrating solution is composed of a CPA ([0158]) and a non-penetrating compound such as a sugar ([0159]). Additionally, Gilad discloses that the CPA concentration in a first of at least two incubations with CPA is between 5-20 wt% and the CPA concentration of a second of at least two incubations with CPA is between 15-30 wt% ([0152]-[0154]). Gilad teaches transferring the embryos to a cryomesh ([0208]).
Gilad does not explicitly describe resultant cryomesh as “a medium packed monolayer of Drosophila embryos with minimal overlap”. However, as stated in the claim interpretation section above, the term “monolayer” is being interpreted to mean a single layer of embryos, in which the embryos can be either sparse or densely packed next to one another; the term “medium packed” is being interpreted to mean anywhere between about 30-70% packed, or in the alternative about 50% or half packed; and the term “minimal overlap” is being interpreted to mean a majority of the embryos do not overlap. Gilad does teach Fig.3A and Fig. 3B, which shows embryos in liquid nitrogen in a monolayer, where about half of the surface is packed with embryos, and although a majority of embryos make contact each other laterally, a majority of the embryos do not appear to overlap one another. Therefore, Fig. 3 of Gilad demonstrates “a medium packed monolayer of Drosophila embryos with minimal overlap”.
With regards to the embryos being “adhered to one or more surfaces of the cryomesh” and “cooling the embryos adhered to the cryomesh by submerging the embryos”, Gilad teaches both freezing and thawing is completed while the embryos are on either a copper mesh or a polycarbonate Nucleopore Whatman paper as required by claims 1 and 18 ([0208]). Additional support for the fact that Gilad cools the embryos adhered to the cryomesh or paper by submerging the embryos in liquid nitrogen is found in Fig. 3 and [0231], which states “FIG. 3 shows vitrified embryos on a Nucleopore Whatman paper in liquid nitrogen after the described peeling and CPA loading protocol or without treatment”. This explanation of Fig. 3 definitively supports that the method of Gilad includes submerging the embryos while still attached to either the mesh or the paper. Therefore, Gilad meets the limitation of cooling the embryos adhered to the cryomesh or “thin copper mesh” as described by Gilad ([0208]).
Regarding claim 2, the staging step comprises visually evaluating the stages of embryonic development including the gut morphology ([0043]/[0189]).
Regarding claim 3, the staging includes incubating the embryo until it has developed to a stage where head involution and dorsal closure has been completed ([0013]).
Regarding claim 6, the permeabilizing step comprises the use of D-Limonene and heptane ([0020]/[0024]).
Regarding claim 7, the CPA solution is comprised of ethylene glycol, propylene glycol, or DMSO ([0053]).
Regarding claim 9, the excess cryoprotective solution is removed ([0222]). Although Gilad does not teach that the excess liquid is removed through wicking, wicking is a method of excess liquid removal well known in the art and is addressed below by Steponkus.
Regarding claim 10, the method also comprises rewarming the embryos ([0208]) in a buffer, which unloads the CPA, and incubating them in a medium ([0032]/[0208]). One of ordinary skill in the art would reasonably read the description in para. 0208 to mean that the rewarming step is completed while the embryos are on the copper mesh, since Gilad does not state removing them to freeze and/or thaw the embryos as required by claim 10 ([0208]).
Regarding claims 11-12, Gilad teaches that the buffer used to rewarm the embryos is Schneider’s medium with trehalose until hatching ([0208]). Gilad meets the limitation of rewarming the embryos in Schneider’s medium for 8 to 24 hours to form larvae because Gilad discloses rewarming in said medium until hatching, i.e. forming larvae.
Regarding claim 13, Gilad teaches the method can be performed with various genetic lines (i.e. wild type or mutant) of different species of flies, including Drosophila ([0066]).
Although Gilad does not teach incubating the embryos at about 20°C for 22 hours as recited in claim 4, one of ordinary skill in the art would recognize that the incubation time and temperature are result effective variables and that the amount of time and the temperature of incubation would be matter of routine optimization as evidenced by Kuntz. Kuntz teaches that Drosophila embryogenesis scales uniformly dependent of temperature. Therefore, depending on the desired incubation time the temperature can be adjusted accordingly (see figure 3).
Gilad does not teach that the cooling rate is greater than 40,000°C/min as required by claim 1, nor that the warming rate is greater than about 100,000°C/min as required by claims 10 and 19.
However, Mazur teaches about the typical cooling and warming rates for drosophila embryos. Mazur teaches that the cooling rates for the drosophila embryos are 110,000 +/- 13,000°C/min as required by claim 1 (pg. 7, column 1, para. 2). Further, Mazur teaches that the warming rates for drosophila are 120,000 +/- 15,000°C/min as required by claims 10 and 19 (pg. 7, column 2, para 1). Finally, Mazur teaches that very high warming rates are critical to survival and that no embryos survive when warmed at a rate of 2,000°C/min (abstract).
One of ordinary skill in the art at the time of the effective filling date of the instant application would find it obvious to combine the method of cryopreserving taught by Gilad with the cooling/warming rates taught by Mazur. One of ordinary skill in the art would be motivated to make this combination because Mazur teaches that very high warming rates are critical to survival and that no embryos survive when warmed at a rate of 2,000°C/min (abstract). One of ordinary skill in the art would have a reasonable expectation of success when combining Gilad with Mazur because Mazur is achieving these cooling and warming rates using liquid nitrogen as taught in Gilad.
With regards to claim 14, Gilad teaches the method can be performed with various genetic lines (i.e. wild type or mutant) of different species of flies, including Drosophila ([0066]).
Gilad does not explicitly teach applying the cryopreservation method to a mutant strain which contains a single nucleotide polymorphism mutation on the X chromosome that improves survival rates after cryopreservation.
However, Gilad does teach that the method can be applied to any Drosophila strain, including a mutant strain and it would be obvious to a skilled artisan at the effective filling date of the instant invention to apply the method of cryopreservation to an embryo from a fly or Drosophila with any genetic background. This would include discovered and undiscovered wild type or mutant strains, further including mutants with cold resistant mutations which aid them in the survival of cryopreservation. One of ordinary skill in the art would be motivated to do this because Gilad teaches that the method can be performed with various genetic lines ([0066]). Further, one of ordinary skill in the art would have a reasonable expectation of success when applying the method of Gilad to a mutant strain because the method is applicable to not only Drosophila, but other various fly species therefore it would be reasonable that the method is effective for mutant drosophila. Thus, the claim is obvious over Gilad.
Gilad teaches that the embryos are dehydrated, but does not teach that they are dehydrated at a temperature lower than room temperature as required by claims 1 and 18. Gilad teaches a method of dechorionating the embryos using bleach, but Gilad does not teach that the concentration of the bleach is 50% as required by claim 1 and 18. Gilad does not teach that dechorionating the embryos for a period of about 2-4 minutes as required by claim 1. Gilad does not teach that the excess cryoprotective solution is removed though wicking as required by claim 9.
However, Steponkus teaches a similar method to that of Gilad, where Drosophila are both dehydrated and dechorionated, in a procedure for the cryopreservation of Drosophila.
Regarding claims 1 and 18, Steponkus teaches that the embryos are dehydrated using 0°C solution and kept on ice during the process (i.e. dehydrated at a temperature lower than room temperature) (pg. 380, Dehydration, last para). Steponkus teaches that the chorion of Drosophila can be removed by a 2.5 min exposure to 50% bleach (Dechorionation, lines 10-11).
Regarding claim 9, Steponkus teaches that the excess cryopreservation solution is removed though wicking with a Kimwipe (pg. 380, last two para).
It would be obvious to one skilled in the art to apply the concentration of bleach for dechorionation taught by Steponkus with the cryopreservation method taught by Gilad and Mazur to arrive at the instant invention. A skilled artisan would be motivated to make this combination because the method disclosed was able to cryopreserve embryos and 45% of them developed into fertile adults (Summary, line 4). One skilled in the art would have a reasonable expectation of success when employing the 50% concentration of bleach because Steponkus teaches the method of dechorionating using Drosophila as well. Thus, Gilad, Mazur, and Steponkus render the claims obvious.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the effective time of filing of the invention, especially in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed 12/16/2025 have been fully considered but they are not persuasive.
Applicant argues (Remarks, pg. 6, last para spanning pg. 7) that the claims have been amended to include the limitation that the embryos form “a medium packed monolayer of Drosophila embryos with minimal overlap”. Applicant argues “as such, a medium packed layer is one in which the embryos are not clumped on the cryomesh and for example occupy 30% of the total mesh area, with minimal overlap [0145]” and continues that Gilad does not disclose this limitation because only a few embryos are demonstrated and the instant invention can place more than 1700 embryos on a 2 cm x 2 cm cryomesh. Further, Applicant argues “the instant specification references that the clumps are broken up which is not disclosed in Gilad”.
In response, this argument is not found persuasive. Gilad does not explicitly describe resultant cryomesh as “a medium packed monolayer of Drosophila embryos with minimal overlap”. However, as stated in the claim interpretation section above, the term “monolayer” is being interpreted to mean a single layer of embryos, in which the embryos can be either sparse or densely packed next to one another; the term “medium packed” is being interpreted to mean anywhere between about 30-70% packed, or in the alternative about 50% or half packed; and the term “minimal overlap” is being interpreted to mean a majority of the embryos do not overlap. Gilad does teach Fig.3A and Fig. 3B, which shows embryos in liquid nitrogen in a monolayer, where about half of the surface is packed with embryos, and although a majority of embryos make contact each other laterally, a majority of the embryos do not appear to overlap one another. Therefore, Fig. 3 of Gilad demonstrates “a medium packed monolayer of Drosophila embryos with minimal overlap”.
Additionally, in response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies are not recited in the rejected claim(s). Applicant relies on an example taken from the specification regarding a medium packed layer being about 30% occupied, which is not recited in the claims. Applicant relies on a volume of more than 1700 embryos on a 2 cm x 2cm cryomesh, which is not recited in the claims. Applicant relies on the instant specification which references a step “that the clumps are broken up which is not disclosed in Gilad”, and which is not recited in the claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). As described in the 112(b) rejections presented above, Applicant may amend claims 1 and 18 to include that the monolayer of embryos occupies about 30% of the total mesh area as recited in the specification, if desired. Applicant may amend claims 1 and 18 to include that the method steps require brushing the clumps of embryos after permeabilizing to break embryo clumps into individual embryos with no overlap as recited in the specification and demonstrated in Fig. 10B, if desired.
Applicant also argues (Remarks, pg. 6, last para spanning pg. 7) that the images of Gilad contain glare and it is not possible for one of ordinary skill to differentiate between glare and what might be ice; and concludes that the images of Gilad appear to have ice which would teach away from the method of Gilad.
In response, the argument is not found persuasive. The images of Gilad are presented in black and white and contain some amount of glare, however the embryos are visible. It is not clear how one of ordinary skill might conclude that Gilad teaches away from the instant method based on the presence of ice, if they are they are unable to differentiate between glare and ice in the images. Therefore, the argument is not found persuasive.
Applicant argues (Remarks, pg. 7, last para spanning pg. 8) that the claims have been amended to require a CPA concentration of less than 40% and Gilad teaches a step wise incubation with increasing concentrations of CPA which go beyond 50% CPA.
In response, this argument is not found persuasive. Gilad discloses that the CPA concentration in a first of at least two incubations with CPA is between 5-20 wt% and the CPA concentration of a second of at least two incubations with CPA is between 15-30 wt% ([0152]-[0154]). The additional incubations of Gilad are alternative embodiments and therefore Gilad meets the claim. Therefore, the argument is not found persuasive. Applicant may amend the claim to require only a single CPA incubation period, if desired.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CONSTANTINA E STAVROU whose telephone number is (571)272-9899. The examiner can normally be reached M-F 8:00-5:00.
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CONSTANTINA E. STAVROU
Examiner
Art Unit 1632
/ANOOP K SINGH/Primary Examiner, Art Unit 1632