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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/09/2026 has been entered.
Claim Status
1. The amendment filed 02/09/2026 has been entered. Claims 1, 3, 5, 6, and 10 – 13 remain pending and are under consideration. Claims 4, 24, and 25 have been cancelled.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 6, 10 – 15, 18 – 21, 24 – 25, and 35) and KDEL (SEQ ID NO:1) in the reply filed on 06/27/2025 is acknowledged.
3. Claims 26 – 27 and 57 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 06/27/2025.
Priority
4. This application is a National Stage application under 35 U.S.C. § 371 of PCT/US2021/028340, filed 04/21/2021, which claims the benefit of U.S. Patent Application No. 63/013,453, filed 04/21/2020.
Withdrawn Claim Objections
5. The objection to claim 24 is rendered moot in view of Applicant’s cancellation of the clam.
Withdrawn Claim Rejections
6. The rejection of claim 4, 24, and 25 under 35 U.S.C. 112(b) is rendered moot in view of Applicant’s cancellation of these claims.
7. The rejection of claims 1, 3, 5, 6, and 10 – 13 under 35 U.S.C. 112(b) is withdrawn in view of Applicant’s amendment to the claim to recite “comprising amino acid residues 11 – 232 of SEQ ID NO: 4.
8. The rejection of claims 24 and 25 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of these claims.
9. The rejection of claim 4 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
10. The rejection of claims 1, 3, 5, 6, and 10 – 13 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
Claim Rejections Necessitated by Amendment
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
11. Claims 1, 3, 5, 6, and 10 – 13 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The instant claims encompass a transgenic mouse wherein somatic cells of said mouse comprise a first nucleic acid encoding a polypeptide comprising amino acid residues 22-232 of SEO ID NO: 4 and an endoplasmic reticulum (ER) retention signal, wherein said first nucleic acid further comprises a promoter sequence followed by a recombinase recognition site followed by an intervening nucleic acid sequence followed by a recombinase recognition site followed by said nucleic acid encoding said, wherein expression of said polypeptide does not occur unless a recombinase excises said intervening nucleic acid sequence via said recombinase recognition sites; and (b) a second nucleic acid comprising an endothelial-specific promoter sequence operably linked to a nucleic acid sequence encoding said recombinase; wherein endothelial cells of said mouse express said polypeptide. Thus the claims are genus claims that broadly encompass any promoter sequence in the first nucleic acid wherein endothelial cells of said mouse express said polypeptide. However, the instant specification fails to describe the entire genus of promoter sequences in the first nucleic acid wherein endothelial cells of said mouse express said polypeptide. Thus, the genus of promoter sequences, which, when present in somatic cells of a transgenic mouse along with the second nucleic acid wherein endothelial cells of said mouse express said polypeptide as claimed, lacks a written description, and as such, there is no indication that Applicants had possession of the claimed invention.
From the specification and drawings, it is clear that Applicants have possession of the CAG promoter in the first nucleic acid and the Cdh5 promoter in the second nucleic acid (page 31, lines 20 – 23; Figure 2A – B; page 32, line 1). However, the claim is not limited to the CAG promoter, yet the claim requires “endothelial cells of said mouse express said polypeptide”. The claims only require broadly a promoter sequence in the first nucleic acid and an endothelial-specific promoter sequence in the second nucleic acid wherein endothelial cells of said mouse express said polypeptide. The specification teaches a promoter sequence can be a naturally occurring promoter sequence or a recombinant promoter sequence or a synthetic promoter sequence or a constitutively active promoter sequence or a regulated promoter sequence and examples include a CAG promoter sequence, a SV40 promoter sequence, a CMV promoter sequence, a UBC promoter sequence, a EF1A promoter sequence and a PGK promoter sequence (page 18, lines 19 – 30).
The specification lacks sufficient variety of species of promoter sequences in the first nucleic acid to reflect this variance in the genus since the specification provides only one example of the CAG promoter in the first nucleic acid sequence with the Cdh5 promoter in the second nucleic acid sequence wherein endothelial cells of said mouse express said polypeptide. The MPEP states that written description for a genus can be achieved by a representative number of species within a broad generic. It is unquestionable that the claims are broad generics, with respect to all of the potential species of promoter sequences that may be in the first nucleic acid. The possible variations of extracts are limitless with potentially thousands of sequences. The purpose of the written description requirement is to ensure that the inventor had possession, as of the filing date of the application, of the specific subject matter claimed by them. A patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that the inventor invented the claimed invention. Thus, an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations," and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention."
In the instant case, the breadth of the genus of promoter sequence in the first nucleic acid, lacks a written description. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. In this case, the specification broadly recites promoter sequences to include natural, recombinant, and synthetic promoter sequences and only teaches CAG as the promoter sequence in the first nucleic acid and the Cdh5 promoter in the second nucleic acid. The skilled artisan cannot envision the sequences of all of the promoter sequences that are encompassed by the claims, and therefore, conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method.
Accordingly, in the absence of sufficient recitation of distinguishing identifying characteristics, the specification does not provide adequate written description of the recited genus. Thus, the written description requirement has not been satisfied.
Applicant is reminded that Vas-Cath makes clear that the written description of 35 U.S.C. 112 is severable from its enablement provision [see p. 1115].
Claim Rejections - 35 USC § 112
12. Claims 1, 3, 5, 6, and 10 – 13 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a transgenic mouse wherein somatic cells other than endothelial cells of said mouse comprise a first nucleic acid encoding a polypeptide comprising amino acid residues 22-232 of SEO ID NO: 4 and an endoplasmic reticulum (ER) retention signal, wherein said first nucleic acid further comprises a CAG promoter sequence followed by a recombinase recognition site followed by an intervening nucleic acid sequence followed by a recombinase recognition site followed by said nucleic acid encoding said, wherein expression of said polypeptide does not occur unless a recombinase excises said intervening nucleic acid sequence via said recombinase recognition sites; and (b) a second nucleic acid comprising an endothelial-specific promoter sequence operably linked to a nucleic acid sequence encoding said recombinase; wherein endothelial cells of said mouse express said polypeptide. Thus the claims are genus claims that broadly encompass any promoter sequence in the first nucleic acid wherein endothelial cells of said mouse express said polypeptide, does not reasonably provide enablement for any promoter sequence in the first nucleic acid sequence. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (a) the breadth of the claims, (b) the nature of the invention, (c) the state of the prior art, (d) the level or ordinary skill in the art, (e) the level of predictability in the art, (f) the amount of direction provided by the inventor, (g) the existence of working examples, and (h) the quantity of experimentation needed to make use of the invention based on the content of the disclosure. While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
(a) The breadth of the claims: The claims broadly recite “promoter sequence” in the first nucleic acid and therefore the claims read on any promoter sequence. Consequently, the breadth of the claims is expansive.
It is noted that the instant rejection is based on absence of enabling disclosure for any “promoter sequence” in the first nucleic acid along with recombinase recognition sites and an intervening sequence between the recombinase recognition sites while requiring an endothelial-specific promoter sequence in the second nucleic acid and wherein endothelial cells of said mouse express said polypeptide. Thus, the claim encompasses promoter sequences in the first nucleic acid in which endothelial cells of said mouse would not express said polypeptide.
(b) The nature of the invention: The nature of the invention is transgenic non-human animals that secrete tagged molecules from a particular tissue (page 1, lines 7 – 10 and 20 – 30; page 2, lines 1 – 3).
(c) The state of the prior art:
The state of the art teaches that Cre is a site-specific recombinase that can turn on a silenced reporter gene or transgene which is usually achieved through Cre-mediated excision of a “stop” sequence of the reporter (trans)gene. Cre recombinase recognizes LoxP sites and deletes the sequence lying in between LoxP sites, leaving behind a single LoxP site and turns on expression of the gene located downstream of the LoxP site. When Cre and reporter (trans)gene are present in the same target cell, Cre-mediated recombination of the reporter (trans)gene leads to constitutive reporter expression. The reporter (trans)gene once recombined in a target cell has to remain expressed at least in every cell type of the tissue or organ under assessment which is often accomplished using the endogenous mouse ROSA locus which is ubiquitously expressed with or without CAG sequences. Cell type- and/or tissue-specific promoters can be genetically modified to drive Cre expression. (Aquila, Iolanda, et al. Pharmacological research 127 (2018): 116-128; page 117, right col. para. 3; Figure 2; page 119, left col. para. 1 – 3)
The state of the art does not appear to provide any evidence that endothelial cells comprising a first nucleic acid comprising an endothelial-cell specific promoter followed by LoxP sites flanking an intervening sequence and a second nucleic acid comprising an endothelial-cell specific promoter operably linked to Cre recombinase would still comprise the intervening sequence flanked by LoxP sites following recognition and deletion by Cre recombinase. Thus, the state of the art teaches an endothelial cell comprising nucleic acids encoding Cre and LoxP sites flanking an intervening sequence would not comprise the LoxP flanked intervening sequence.
(d) The level skill in the art:
The level of skill in the art of creating transgenic mice is high, as an artisan in this art needs specialized knowledge such as a postgraduate degree (Ph.D. and/or M.D.) given the complex nature of the mouse transgenics.
(e) The level of predictability in the art:
The predictability of endothelial cells comprising the claimed first and second nucleic acid where the promoter sequence is any sequence and wherein endothelial cells of said mouse express said polypeptide is low given the prior art teaches that Cre recombinase would delete the claimed intervening sequence and leave one LoxP site.
(f) The amount of direction provided by the inventor:
The specification shows that endothelial cells do not comprise the intervening sequence or both loxP sites when the promoter sequence in the first nucleic acid is CAG in Figure 2A.
(g) The existence of working examples:
The specification teaches a transgenic mouse comprising a ER-BioIDHA construct containing a CAG promoter that was crossed with Cdh5-Cre mice to delete the loxP-Stop-loxP cassette (Example 1, page 28 – 32).
Thus, the specification provides sufficient teachings only for the enablement of somatic cells other than endothelial cells comprising the claimed first nucleic acid wherein the promoter sequence is CAG.
(h) The quantity of experimentation needed to make use of the invention based on the content of the disclosure:
The amount of experimentation would be undue because it would require determining how to maintain the first nucleic acid containing the intervening sequence flanked by loxP sites in endothelial cells wherein the endothelial cells express said polypeptide and what promoter sequence would allow for maintaining the claimed first and second nucleic acids wherein endothelial cells express said polypeptide. This is because one cannot extrapolate between the working example in the specification and any promoter sequence in the first nucleic acid and since the prior art teaches that Cre deletes the intervening sequence flanked by loxP sites. Thus, the full scope of claim 1 is not enabled by the disclosure.
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 (i.e., changing from AIA to pre-AIA ) 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
13. Claim(s) 1, 3, 5, 6, and 10 – 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhou (Zhou, Pingzhu, et al. Proceedings of the National Academy of Sciences 110.38 (2013): 15395-15400; previously cited), hereinafter Zhou as evidenced by Jackson Laboratory (Strain 022386. Jackson Laboratory, https://www.jax.org/strain/022386, Retrieved 11/14/2025; previously cited), hereinafter Jackson Laboratory in view of Croall (Croall PhD, Dorothy E., et al. (2016).), hereinafter Croall in view of Roux (WO2014070227A1; Filed 03/11/2013; Published 05/08/2014; previously cited), hereinafter Roux in view of Driegen (Driegen, Siska, et al. Transgenic research 14.4 (2005): 477-482; previously cited), hereinafter Driegen in view of Bartelle (Bartelle, Benjamin B., et al. Circulation research 110.7 (2012): 938-947; previously cited), hereinafter Bartelle.
Regarding claim 1 and 10, Zhou teaches a transgenic mouse whose somatic cells comprise a nucleic acid comprising a CAG promoter (“promoter sequence” of claim 1) followed by a loxP site (“recombinase recognition site” of claim 1; loxP site of claim 10) followed by an intervening sequence (“intervening sequence” of claim 1) followed by a loxP site (“recombinase recognition site” of claim 1; loxP site of claim 10) followed by Escherichia coli BirA (Figure 1B and F; page 15396, left col.; Figure S1). Zhou teaches BirA expression only in the presence of Flp recombinase (page 15396, left col. para. 2). Zhou does not teach “comprising amino acid residues 22 – 232 of SEQ ID NO: 4” or a nucleic acid encoding an ER retention signal of claim 1.
Regarding claim 1, Zhou teaches a transgenic mouse whose somatic cells comprise a Rosa26frTRAP allele comprising a first nucleic acid comprising a CAG promoter (“a promoter sequence” of claim 1) followed by recombinase site (“recombinase recognition site”) followed by an intervening nucleic acid sequence (“an intervening nucleic acid sequence” of claim 1) followed by a recombinase site (“recombinase recognition site” of claim 1) followed by a sequence encoding E. coli BirA that is a biotin ligase (Figure S1A; page 15396, left col. para. 2). Zhou does not teach the sequence encoding E. coli BirA biotin ligase encodes amino acid residues 22 – 232 of SEQ ID NO: 4 that is a biotin ligase or a nucleic acid encoding an ER retention signal of claim 1. Zhou teaches the transgenic mouse Tie2Cre::Rosa26fsTRAP comprises a second nucleic acid comprising the endothelial-specific promoter sequence Tie2-Cre (“(b)” of claim 1) where endothelial cells express E.coli BirA (page 15396, left col. para. 2; Figure 2, S1E, S2; page 15397, left col. last para.). Zhou teaches E. coli BirA is only expressed when a recombinase excises the intervening nucleic acid sequence (page 15396, left col. para. 2). Therefore, Zhou teaches the somatic cells of the Tie2Cre::Rosa26fsTRAP mouse would comprise the Rosa26frTRAP allele and Tie2-Cre nucleic acid in non-endothelial cells and would comprise the Rosa26frTRAP allele without the recombinase sites or intervening sequence and the Tie2-Cre nucleic acid in endothelial cells, where the endothelial cells express nucleic acid sequences following the recombinase recognition sites that were removed by the recombinase.
Regarding claim 5 and 6, Zhou teaches the Rosa26fs-TRAP mice are available through The Jackson Laboratory stock number 022386 (page 1 of Supporting Information, left col. para. 3) where BirA comprises an HA tag (“peptide tag of claim 5 and “HA tag” of claim 6) as evidenced by Jackson Laboratory (page 2, para. 2 Development).
Regarding claim 10, Zhou teaches in Figure 1B and Figure S1A LoxP recognition sites.
Regarding claim 11, Zhou teaches the nucleic acid comprises a transcriptional stop signal (page 15396, left col. para. 2 – 3 and right col. para. 2; Supporting Information page 1, left col. para. 1).
Regarding claims 12 and 13, Zhou teaches the nucleic acid comprises an intervening sequence that is EGFP-L10 (“intervening sequence”, “polypeptide” of claim 12, “EGFP” of claim 13) (Figure 1B, C; Figure S1A and C; Supporting Information page 1, left col. para. 1).
Zhou does not teach a nucleic acid encoding “comprising amino acid residues 22 – 232 of SEQ ID NO: 4” or an ER retention signal of claim 1 that is KDEL of claim 3. However, Zhou teaches the transcripts in the Tie2Cre::Rosa26fsTRAP mouse (Tie2-TRAP) is a measure of endothelial cell enrichment and is a way to interrogate cell type-specific transcriptomes (page 15397, right col. para. 1 – 4; page 15399, right col. para. 4). Zhou teaches transcriptional profiling is a useful strategy to study development and disease and approaches to isolate RNA from specific cell types or cellular compartment would extend the power of this strategy (Abstract; page 15340, right col. para. 3). Zhou teaches whole-tissue transcript levels represent the average of the distinct cell lineages in the tissue and can result in loss of important information and RNA profiling measures transcript abundance but transcriptional regulation is also an important determinant of gene expression (page 15395, left col. para. 1). Zhou teaches a known limitation of gene expression profiling of whole tissue is that gene expression changes in disease models may occur as a result of a change in the proportion of cell types, or in cells that are not the primary interest of the study (page 15400, left col. para. 2).
Croall teaches in vitro determination of the interactome for calpain2 with proteins from endothelial cells by using calpain2 with a biotin acceptor peptide (calpain2*) that is targeted by the biotin ligase BirA as bait where biotinylated calpain2-endothelial cell protein interactions are captured (page 3, col. 1, panel 1; page 3, col. 2). Croall teaches the reason for choosing endothelial cells to determine calpain2 protein-protein interactions is that defects in formation and repair of blood vessels occur with overexpression of calpastatin, the specific endogenous inhibitor of calpain2 implying that calpain2 contributes to angiogenesis and vascular repair but its roles are undefined (page 3, col. 1, panel 3). Croall teaches calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, although mechanistic details of calpain’s roles are lacking (page 3, col. 1, panel 3). Croall teaches capture of calpastatin and known calpain2 substrates validates the success of the capture strategy (page 3, last col. panel 4). Croall does not teach “comprising amino acid residues 22 – 232 of SEQ ID NO: 4” or an ER retention signal of claim 1 that is KDEL of claim 3. One would have been motivated to combine the teachings of Zhou and Croall in a transgenic mouse with endothelial-specific expression of BirA to determine in vivo protein-protein interactions with calpain2* to complement endothelial cell transcriptomes because Zhou teaches the transcripts in the Tie2Cre::Rosa26fsTRAP mouse (Tie2-TRAP) is a way to interrogate cell type-specific transcriptomes and Croall teaches calpain2 may contribute to angiogenesis and vascular repair but its roles are undefined and calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, but mechanistic details of calpain’s roles are lacking.
Regarding “comprising amino acid residues 22 – 232 of SEQ ID NO: 4” of claim 1, Roux teaches the sequence of A. aeolicus promiscuous biotin protein ligase that comprises amino acid residues 22 – 232 of SEQ ID NO: 4 (Figure 8A; page 49, lines 12 – 17, SEQ ID NO: 15 and 16). Roux teaches the use of this biotin protein ligase for identifying in vivo proximate proteins using the BioID method to identify neighboring and potentially interacting proteins (page 1, lines 30 – 34; page 2, lines 1 – 3; Figure 1; page 5, lines 25 – 33; page 26, lines 27 – 30). Roux teaches the biotin ligase is fused to a protein of interest and then introduced into mammalian cells where it will biotinylate vicinal proteins and biotinylated proteins can then be selectively isolated and identified by conventional methods including mass spectrometry (page 26, lines 30 – 34; page 33, lines 10 – 18). Roux teaches in Example 2 promiscuous A. aeolicus BPL with mutation R40G that improves the BioID method (page 48, lines 1 – 17). Roux teaches elucidation of protein-protein interactions represents a significant barrier to the understanding of complex biological processes (page 1, lines 11 – 12). Roux teaches it has become increasingly clear that the functions of many proteins can only be fully understood in the context of networks of interactions and the description of such networks provides keys to our understanding of disease processes (page 1, lines 12 – 15). Roux teaches affinity-capture complex purification provide powerful tools in the search for new molecular associations but display fundamental limitations because they are commonly assessed in a cellular environment different to that in which they would normally occur (page 1, lines 16 – 22). Roux does not teach an ER retention signal of claim 1 that is KDEL of claim 3. One would have been motivated to combine the teachings of Zhou, Croall, and Roux in a transgenic mouse with endothelial-specific expression of calpain2-biotin protein ligase to determine in vivo protein-protein interactions with calpain2 to complement endothelial cell transcriptomes because Zhou teaches the transcripts in the Tie2Cre::Rosa26fsTRAP mouse (Tie2-TRAP) is a way to interrogate cell type-specific transcriptomes and Croall teaches calpain2 may contribute to angiogenesis and vascular repair but its roles are undefined and calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, but mechanistic details of calpain’s roles are lacking and Roux teaches the BioID method can identify interacting proteins in vivo.
Driegen teaches a transgenic mouse that expresses the BirA gene by insertion into the ROSA26 locus where the BirA protein is expressed in all tissues tested (Abstract; page 478, left col. para. 3 – 5; Figure 1d). Driegen teaches the nucleic acid comprising BirA also comprises an HA-tag and that BirA expression could be detected in all tissues tested (page 478, right col. para. 2; Figure 1d). Driegen teaches the BirA mouse strain is an excellent strain to obtain biotinylation of an endogenous protein as BirA expression is sufficient to biotinylate a protein in mouse tissue with a biotinylation sequence (page 481, right col. para. 3). Driegen teaches the BirA mouse might prove useful in a wide range of studies in which complex interactions need to be studied in the developing or adult animal (page 481, right col. para. 2). Driegen teaches one potential drawback of these BirA transgenic mice is that the proteins that are translated on the endoplasmic reticulum bound ribosomes and are entering the secretory pathway will not be efficiently biotinylated by the BirA protein which is located largely in the cytoplasm (page 481, right col. last para.). Driegen teaches another study has shown that proteins in the secretory pathway can be efficiently biotinylated provided that BirA protein itself is extended with a signal peptide that will direct the protein into the secretory pathway (page 481, right col. last para.). Driegen teaches that expression of the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues (Abstract). Driegen does not teach an ER retention signal that is KDEL claims 1 and 3. One would have been motivated to combine the teachings of Zhou, Croall, Roux, and Driegen in a transgenic mouse with endothelial-specific expression of calpain2-biotin protein ligase to determine in vivo protein-protein interactions with calpain2 to complement endothelial cell transcriptomes because Zhou teaches the transcripts in the Tie2Cre::Rosa26fsTRAP mouse (Tie2-TRAP) is a way to interrogate cell type-specific transcriptomes and Croall teaches calpain2 may contribute to angiogenesis and vascular repair but its roles are undefined and calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, but mechanistic details of calpain’s roles are lacking and Roux teaches the BioID method can identify interacting proteins in vivo and Driegen teaches the BirA mouse might prove useful in a wide range of studies in which complex interactions need to be studied in the developing or adult animal.
Regarding an ER retention signal that is KDEL claims 1 and 3, Bartelle teaches a transgenic mouse comprising a nucleic acid encoding biotin ligase and an ER retention signal that is KDEL (12xBiotag-IRES-BirAER) in somatic cells (Abstract; Figure 1A; page 939, left col. para. 3 and last para; page 940, left col. para. 1; page 941, left col. para. 1 and right col. para. 1 – 3; Online Figure I – II; Supplemental page 1, Expression Constructs). Bartelle teaches expression of Biotag and BirA in the secretory pathway where they could interact and generate a biotinylated membrane protein for targeting with avidinated probes (page 940, left col. para. 1; Figure 1). Bartelle teaches in Figure 1 that the biotin ligase is retained in the ER by the KDEL sequence where it can biotinylate a protein. Bartelle teaches a transgenic mouse comprising a nucleic acid encoding BirA biotin ligase and an ER retention signal (12xBiotag-IRES-BirAER) in somatic cells where BirA is operably linked to Tie promoter (Abstract; Figure 1A; page 939, left col. para. 3 and last para; page 941, left col. para. 1 and right col. para. 1 – 3; Online Figure I – II; Supplemental page 1, Expression Constructs). Bartelle teaches expression of BirA in somatic cells (ear, tail) (Figure 5A, 6, 7; Supplemental page 3, para. 1 – 2; Online Figure 1). Bartelle teaches the mouse allows for studies of vascular biology in the vasculature (Abstract).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Zhou regarding a transgenic mouse comprising a first nucleic acid comprising a promoter followed by a recombinase recognition site followed by an intervening sequence followed by a recombinase recognition site followed by a sequence encoding a biotin ligase and a second nucleic acid comprising a Tie2 promoter followed by a nucleic acid sequence encoding Cre recombinase where the endothelial cells express the biotin ligase with the teachings of Croall regarding determining protein-protein interactions of calpain2 with endothelial cells in vitro with biotinylation with the teachings of Roux regarding determining protein-protein interactions in vivo with BioID with a promiscuous A. aeolicus biotin ligase with the teachings of Driegen regarding biotin ligase transgenic mice to identify in vivo protein-protein interactions with the teachings of Bartelle regarding a transgenic mouse expressing biotin ligase with an ER retention signal from a Tie2 promoter to arrive at the claimed transgenic mouse wherein somatic cells of said mouse comprise a first nucleic acid encoding a polypeptide comprising amino acid residues 22-232 of SEO ID NO: 4 and an endoplasmic reticulum (ER) retention signal, wherein said first nucleic acid further comprises a promoter sequence followed by a recombinase recognition site followed by an intervening nucleic acid sequence followed by a recombinase recognition site followed by said nucleic acid encoding said, wherein expression of said polypeptide does not occur unless a recombinase excises said intervening nucleic acid sequence via said recombinase recognition sites; and (b) a second nucleic acid comprising an endothelial-specific promoter sequence operably linked to a nucleic acid sequence encoding said recombinase; wherein endothelial cells of said mouse express said polypeptide. One would have been motivated to combine the teachings of Zhou, Croall, Roux, Driegen, and Bartelle in a transgenic mouse for transcriptional profiling and proteomic studies to determine the role of calpain 2 in endothelial cells as Zhou teaches transcriptional profiling is a useful strategy to study development and disease and approaches to isolate RNA from specific cell types or cellular compartment would extend the power of this strategy and Croall teaches calpain2 may contribute to angiogenesis and vascular repair but its roles are undefined and calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, but mechanistic details of calpain’s roles are lacking and Roux teaches elucidation of protein-protein interactions represents a significant barrier to the understanding of complex biological processes and Roux teaches affinity-capture complex purification provide powerful tools in the search for new molecular associations but display fundamental limitations because they are commonly assessed in a cellular environment different to that in which they would normally occur and Driegen teaches the BirA mouse might prove useful in a wide range of studies in which complex interactions need to be studied in the developing or adult animal and Driegen teaches one potential drawback of these BirA transgenic mice is that the proteins that are translated on the endoplasmic reticulum bound ribosomes and are entering the secretory pathway will not be efficiently biotinylated by the BirA protein which is located largely in the cytoplasm. One would have a reasonable expectation of success in combining the teachings as Zhou teaches the transcripts in the Tie2Cre::Rosa26fsTRAP mouse (Tie2-TRAP) is a measure of endothelial cell enrichment and is a way to interrogate cell type-specific transcriptomes and Croall teaches capture of calpastatin and known calpain2 substrates validates the success of the capture strategy and Roux teaches the use of this biotin protein ligase for identifying in vivo proximate proteins using the BioID method to identify neighboring and potentially interacting proteins and Driegen teaches the BirA mouse strain is an excellent strain to obtain biotinylation of an endogenous protein as BirA expression is sufficient to biotinylate a protein in mouse tissue with a biotinylation sequence and Bartelle teaches that the biotin ligase is retained in the ER by the KDEL sequence where it can biotinylate a protein.
Applicant’s Arguments/ Response to Arguments
25. Applicant asserts: On page 5, last paragraph, Applicant disagrees with obviousness rejection of the claims at no point do the combinations of cited references teach or suggest that a person having ordinary skill in the art should make or use a transgenic mouse of amended claim 1.
Response to arguments: The prior rejections have been withdrawn in view of the amendment to claim 1. In the new rejection set forth above, the combined teachings of Zhou regarding a transgenic mouse comprising a first nucleic acid comprising a promoter followed by a recombinase recognition site followed by an intervening sequence followed by a recombinase recognition site followed by a sequence encoding a biotin ligase and a second nucleic acid comprising a Tie2 promoter followed by a nucleic acid sequence encoding Cre recombinase where the endothelial cells express the biotin ligase with the teachings of Croall regarding determining protein-protein interactions of calpain2 with endothelial cells in vitro with biotinylation with the teachings of Roux regarding determining protein-protein interactions in vivo with BioID with a promiscuous A. aeolicus biotin ligase with the teachings of Driegen regarding biotin ligase transgenic mice to identify in vivo protein-protein interactions with the teachings of Bartelle regarding a transgenic mouse expressing biotin ligase with an ER retention signal from a Tie2 promoter would motivate one of ordinary skill in the art to arrive at the claimed transgenic mouse for transcriptional profiling and proteomic studies to determine the role of calpain 2 in endothelial cells because Zhou teaches transcriptional profiling is a useful strategy to study development and disease and approaches to isolate RNA from specific cell types or cellular compartment would extend the power of this strategy and Croall teaches calpain2 may contribute to angiogenesis and vascular repair but its roles are undefined and calpains are implicated in the response of endothelial cells to a variety of hormonal and/or cytokine signals and potentially play an integrative role, but mechanistic details of calpain’s roles are lacking and Roux teaches elucidation of protein-protein interactions represents a significant barrier to the understanding of complex biological processes and Roux teaches affinity-capture complex purification provide powerful tools in the search for new molecular associations but display fundamental limitations because they are commonly assessed in a cellular environment different to that in which they would normally occur and Driegen teaches the BirA mouse might prove useful in a wide range of studies in which complex interactions need to be studied in the developing or adult animal and Driegen teaches one potential drawback of these BirA transgenic mice is that the proteins that are translated on the endoplasmic reticulum bound ribosomes and are entering the secretory pathway will not be efficiently biotinylated by the BirA protein which is located largely in the cytoplasm.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632